Following UV-B irradiation, wrinkles appear on the dorsal skin (arrowheads) shown in 2 animals (tail area marking) compared with intact untreated mice to the left.
A, A control animal showing numerous bumps (arrowhead). B, UV-B irradiation elicits formation of coarse wrinkles and disappearance of bumps. C, Peptide treatment demonstrates eradication of wrinkles and normal distribution of bumps.
A, Graphical representation of mean (SD) Rz values in different groups. For an explanation of Rz value, see the Replica Preparation subsection in the Methods section. B, A bar graph showing mean epidermal thickness in control, UV-irradiated, and irradiated animals exposed to topical treatments. Error bars indicate SD. C, Proliferating cell nuclear antigen (PCNA) index in control, UV-irradiated, and irradiated animals exposed to topical treatments. Error bars indicate SD. PCNA index is the number of positive nuclei divided by the total number of keratinocyte nuclei multiplied by 100. AO indicates antioxidant; Est, estrogen; RAD, irradiated; RAD-long, irradiated long; Pep, peptide; and RA, retinoic acid.
A, Skin section from an untreated, intact animal stained with trichrome procedure. B, Irradiated skin section stained with same procedure showing widening of epidermis, and proliferation of follicles. C, Irradiated animal topically treated with estrogen showing further expansion of the epidermis and exaggerated cellular layers. D, Irradiated skin topically treated with peptide cream showing almost normal appearance. E and F, Immunostained proliferating cell nuclear antigen–positive cells in a control animal restricted to the basal layer (arrowhead [E]), which proliferated following irradiation (arrowhead [F]). EPI indicates epidermis; and DER, dermis.
Bhattacharyya TK, Pathria M, Mathison C, Vargas M, Thomas JR. Cosmeceutical Effect on Skin Surface Profiles and Epidermis in UV-B–Irradiated Mice. JAMA Facial Plast Surg. 2014;16(4):253-260. doi:10.1001/jamafacial.2013.2582
Copyright 2014 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.
These data may be useful for developing guidelines for clinicians and the general population related to the reversal of photoaging effects on the aging face damaged by solar radiation.
To investigate antiaging effects of 4 commercially available topical agents on the dorsal skin in photoaged hairless mice.
Design and Setting
Animal study at an academic medical center. Animals comprised 56 female Skh-1 hairless mice (6-8 weeks old). Skin samples were collected from nonirradiated intact mice (control), mice irradiated with UV-B for 8 weeks, mice irradiated with UV-B and then exposed to a topical cosmeceutical applied for 5 weeks, and UV-B-irradiated mice not exposed to cosmeceuticals and retained for 5 weeks until the end of the experiment.
The mice were exposed to UV-B light 3 times a week for 2 months, followed by topical application of a peptide, antioxidant, estrogen, and retinoic acid agent for 5 weeks.
Main Outcomes and Measures
Surface features such as wrinkling were analyzed from replicas along with histomorphometric determination of epidermal thickness, sebocyte counts, and immunohistochemical study of proliferating cell nuclear antigen (PCNA).
Exposure to UV-B induced significant wrinkle formation after 13 weeks, which was attenuated with treatments with a peptide cream, antioxidant mixture, and estrogen cream (mean [SD] Rz values: control [C], 60.7 [19.0]; irradiated [RAD], 51.8 [15.9] [P < .001]; irradiated-long [RAD-long], 86.0 [28.3] [P = .01]; antioxidant [AO], 45.2 [13.2]; peptide, 63.4 [18.8], estrogen, 64.6 [21.2]; retinoic acid [RA], 73.9 [28.5]; RAD-long vs C [P = .01], vs RAD [P < .001], vs estrogen [P = .04], vs peptide [P = .02], vs AO [P<.001], vs RA [P = .25]. There was a trend of reversal of irradiation-induced augmentation of epidermal thickness in animals treated with the peptide and AO (mean [SD] epidermal width: C, 21.0 [2.2] μm; RAD, 41.3 [7.0] μm [P < .001]; RAD-long, 39.1 [11.0] μm [P = .006]; AO, 37.3  μm [P < .001]; peptide, 33.9 [3.8] μm [P = .01]; estrogen, 59.2 [9.2] μm [P = .003]; RA, 52.4 [8.7] μm [P < .001]). Retinoic acid augmented epidermal width and sebocyte counts (mean [SD] sebocyte data [number per gland]: C, 9.4 [2.0]; RAD, 11.69 [1.5] [P < .001]; RAD-long, 6.5 [1.3] [P = .73]; peptide, 7.2 [1.7] [P = .03]; estrogen, 4.1 [0.9] [P < .001]; AO, 7.2 [1.7] [P = .06]; RA, 11.0 [1.4] [P = .01]). Estrogen cream was effective in restoring surface features but enhanced thickness of epidermis in irradiated specimens. All groups had a higher PCNA index score except for peptide treatment, which brought it down to the control level (mean [SD] PCNA index values: C, 17.3 [1.5]; RAD, 32.4 [6.8] [P < .001]; RAD-long, 34.0 [6.1] [P < .001]; AO, 62.1 [3.5] [P = .01]; peptide, 20.1 [6.3] [P < .001]; estrogen, 56.8 [10.0] [P < .001]; RA, 35.2 [10.2] [P < .001]).
Conclusions and Relevance
Of the 4 cosmeceuticals tested within this experimental period, peptide cream and antioxidant mixture were the most effective overall in reversing photoaging effects; retinoic acid was the least effective of these topical agents.
Level of Evidence
Exposure to UV-B radiation is associated with profound changes in biomolecules in skin resulting in photodamage that involves a cascade of metabolic, enzymatic, and phenotypic alterations, such as wrinkle formation.1 Among baby boomers, there is great concern about the effects of age-induced changes of the human face, and apart from surgical interventions for improvement of the facial skin, topical treatment with various antiaging substances on the skin damaged by sun exposure is a subject of popular interest. Antiaging topical products are a large segment of the cosmetic skin care industry and are widely used for reversal of some of the damage caused by solar radiation.2 An array of newer products that appeal to consumers emerges frequently in the commercial market; experimental studies on such products that can supposedly reverse degenerative skin changes emanating from exposure to solar radiation can be helpful to clinicians and consumers.3 Among such products, notable benefits have been observed with respect to retinoids, peptides, and antioxidants (AOs).4
We have reported histological and morphometric responses to such agents5,6 in the intact, nonirradiated hairless mouse, which has been a favorite model for photoaging studies during the last few decades.7 In this investigation, these mice were exposed to UV-B radiation to induce photodamage, followed by topical application of 4 commercially available antiaging agents. The surface profile of skin samples was analyzed and epidermal width and cell proliferation were measured to compare the relative efficacy of these agents on these experimental parameters.
All animal procedures were performed per approved animal protocol from the institutional animal welfare committee of the University of Illinois at Chicago. Skh-1 hairless mice (6-8 weeks old) obtained from Charles River Laboratories were used for this experiment. The following groups were set up (8 animals in each group): (1) intact nonirradiated mice (control); (2) irradiated (RAD) mice; (3) mice irradiated for 8 weeks and then held for another 5 weeks without treatment (RAD-long); and irradiated mice followed by topical application of (4) AO mixture, (5) estrogen, (6) peptide cream, and (7) retinoic acid (RA [tretinoin cream]). The experimental sequence was as follows: exposure to UV-B radiation for 8 weeks (control and RAD groups were humanely killed at the end of 8 weeks), followed by 5 weeks of topical treatment (AO, estrogen, peptide, and RA) given to irradiated mice and 5 weeks of no topical treatment (RAD-long, AO, estrogen, peptide, and RA groups were humanely killed at the end of the 13th week).
Skin specimens were collected from nonirradiated intact controls and irradiated animals at the end of 8 weeks for replica preparation and histologic analysis. Dorsal skin areas from the RAD-long group and cream-treated areas (from AO, estrogen, peptide, and RA groups) were collected at the end of the 13th week for making silicon replicas and histologic slide preparations.
The following commercially available cosmeceuticals were applied to tattooed areas of the irradiated dorsal skin 3 times a week: (1) AO group, C E Ferulic (combination AOs with l-ascorbic acid, α-tocopherol, and ferulic acid; SkinCeuticals); (2) peptide group, Replenix peptide cream (Replenix Facial firming therapy with acetyl hexapeptide-8, acetyl dipeptide-1, palmitoyl tripeptide-3, Macrocystis pyrifera extract; Topix Pharmaceuticals Inc); (3) estrogen group, estrogen cream (Estriol-M 0.3% facial serum; Madison Pharmacy Associates Inc); (4) RA group, Renova (tretinoin cream 0.05%; Ortho Dermatological).
We used sunlamps from a Research Radiation Unit (Daavlin) that emit approximately 80% UV-B radiation in the range of 280 to 340 nm, with a peak emission at 314 nm. In preliminary experiments, animals were irradiated with single doses of UV-B (60, 70, 80, 90, 100, 110, 120, 180, and 240 mJ/cm2) to determine the minimum erythematous dose (approximately 90 mJ/cm2 under the experimental condition), and the data were compared with nonirradiated control animals. Signs of erythema (redness), edema, and blistering were visually observed, photographed, and tabulated from day 1 to day 5 after irradiation. Erythema was graded following standard scales established by other authors for this species.8 The details have been given elsewhere.9 UV-B radiation was applied to the back of the mice, which were freely moving in cages 3 times a week for 8 weeks. The amount of radiation was progressively increased from 30 mJ/cm2 per exposure at week 1 to 90 mJ/cm2 per exposure at week 8 (30, 60, 70, and 80 mJ for 4 weeks and 90 mJ for a further 4 weeks).
The animals were humanely killed on the fourth or fifth day after conclusion of the treatment period. At autopsy, silicon replicas of skin samples were prepared using resin and rings for the evaluation of skin microrelief by Standard Replica Analysis (BIONET protocol published by CuDerm Corp). The replica analysis was performed with specialized equipment (eg, personal computer, video, OPTIMAS, Excel [Microsoft Corp], and Statistica software [StatSoft Inc]). Wrinkle analysis was done with traditional surface roughness statistics (Rz, Ra, and FNum).10 Rz and Ra are optical counterparts of classic “stylus” roughness texture parameters and increase with greater roughness. The surface of hairless mouse skin also has numerous nodular projections (described here as “bumps”), and the height, diameter, and count of these structures that appear as bumps on skin surface were recorded. For simplification, analysis of wrinkles in this study was based on 2 parameters, ie, Rz and number of bumps.
Compound treated, irradiated, and untreated intact areas of dorsal skin were excised, flattened on filter paper, and immersed in Bouin-Hollande fluid for 48 hours. Contiguous areas used for preparing silicon replicas were also fixed in a 10% neutral-buffered formalin solution to generate a second set of slides for study. Subsequently, the samples were dehydrated and processed by paraffin embedding. Paraffin sections (5 μm in thickness) were stained with hematoxylin-eosin–phloxine sequence and trichrome procedures. Sections were also immunostained for epidermal proliferating cell nuclear antigen (PCNA). Deparaffinized sections were pretreated with EDTA, citrate, or liberate antibody binding solution (L.A.B. Solution; Polysciences Inc) for heat-induced epitope retrieval. Sections were treated with a blocking solution provided in a commercially available staining kit (Zymed Laboratories Inc) that uses a biotinylated PCNA monoclonal antibody, streptavidin peroxidase, as a signal generator and diaminobenzidine as the chromogen. After reaction, the sections were lightly stained with hematoxylin, resulting in adequate contrast for counting.
The microscope ocular lens was fitted with a square lattice grid. For computing PCNA-positive cells, epidermal nuclei present within the grid were counted at ×45 magnification, avoiding follicular areas. Nuclei with intense brown staining or sharply stained granules were considered positive for immunoreactivity, and nuclei with diffuse and faint staining were disregarded. The PCNA index is the number of positive nuclei divided by the total number of keratinocyte nuclei multiplied by 100.5 For morphometric analysis, epidermal thickness was manually measured with a calibrated ocular micrometer scale introduced into the microscope eyepiece. Linear measurements were made from the basement membrane to the end of the granular layer in interfollicular sites.11 All morphometric observations are based on data collected from 7 to 8 animals. Slide readings were performed in blinded fashion. Results are expressed as mean (SD). Analysis of variance was used to detect overall difference between means of the 7 groups. The Fisher least significant difference test was performed for post hoc analysis of pairwise comparisons to detect differences between group means, and the level of significance was assigned at P < .05. Statistical tests were performed using commercially available software, ie, Excel (Microsoft Corp) and SPSS version 20.0 (SPSS Inc)
Table 1 illustrates quantitative data on replica surface features (Rz values and number of bumps on the skin surface), and histological features of the hairless mouse epidermis in control, UV-treated, and UV-treated skin with topically administered cosmeceutical agents (AO, peptide, estrogen, and RA). Wrinkle formation was visibly apparent from 6 to 7 weeks of UV-B treatment (Figure 1), but Rz values were significantly altered only in the RAD-long group. In other words, statistically significant wrinkle formation in untreated skin resulted after 13 weeks (Figure 2). This wrinkle formation was reversed by topical agents. Peptide, AO, and estrogen reduced wrinkle formation in 5 weeks. Roughness parameter Rz values were higher in the UV-irradiated RAD-long group (13 weeks) compared with the RAD group (8 weeks) (mean [SD] RZ values: control, 60.7 [19.0]; RAD, 51.8 [15.9]; RAD-long, 86.0 [28.3]; AO, 45.2 [13.2]; peptide, 63.4 [18.8], estrogen, 64.6 [21.2]; RA, 73.9 [28.5]). The post hoc Fisher least significant difference test on Rz values showed the following significant differences: RAD-long compared with the control (P = .01), irradiation (P < .001), AO (P < .001), estrogen (P = .04), and peptide (P = .02) groups; no significant difference was observed for the RA group (Figure 3A). Interestingly, the number of bumps showed a reverse trend. The number was the smallest in RAD-long group; the 4 treatments yielded higher values (mean [SD] bump number: control, 13.0 [3.9]; RAD, 9.3 [6.7] [P = .11]; RAD-long, 5.3 [3.7] [P < .001]; AO, 15.0 [3.9] [P = .04]; peptide, 7.8 [3.6] [P = .03]; estrogen, 7.1 [5.3] [P = .01]; RA, 6.5 [3.7] [P = .007]). Table 2 gives a qualitative grading of changes in experimental parameters from all groups.
The epidermal width, which showed larger values in irradiated animals, was reversed following peptide and AO treatment. Curiously, both estrogen and RA treatment augmented the thickness to a level higher than after irradiation (Figure 3B) (mean [SD] epidermal width: control, 21.0 [2.2] μm; RAD, 41.3 [7.0] μm [P < .001]; RAD-long, 39.1 [11.0] μm [P = .006]; AO, 37.3  μm [P < .001]; peptide, 33.9 [3.8] μm [P = .01]; estrogen, 59.2 [9.2] μm [P = .003]; RA, 52.4 [8.7] μm [P < .001]). Epidermal PCNA values (Figure 3C) were heightened following irradiation (RAD and RAD-long groups), were higher in AO and estrogen groups than in the RAD and RAD-long groups, and similar to the mean values in the irradiated group treated with RA. Only peptide treatment brought the index to near-control levels. The AO group animals showed somewhat contradictory results because despite regression in epidermal thickness, the PCNA multiplication rate was still high (PCNA index values: control, 17.3 [1.5]; RAD, 32.4 [6.8] [P < .001]; RAD-long, 34.0 [6.1] [P < .001]; AO, 62.1 [3.5] [P = .01]; peptide, 20.1 [6.3] [P < .001]; estrogen, 56.8 [10.0] [P < .001]; RA, 35.2 [10.2] [P < .001]).
Histological changes are illustrated in Figure 4. UV-B irradiation alone induced epidermal thickening and proliferation of sebaceous follicles (Figure 4A and B). The RAD group animals had a higher number of sebocytes per gland demonstrating proliferation following irradiation. The number declined in RAD-long group. Peptide and estrogen treatment brought down this number, and AO did not have any noticeable effect, whereas RA caused an increased count over control values. The results were statistically significant (mean [SD] sebocyte data [number per gland]: control, 9.4 [2.0]; RAD, 11.69 [1.5] [P < .001]; RAD-long, 6.5 [1.3] [not significant]; peptide, 7.2 [1.7] [P = .03]; estrogen, 4.1 [0.9] [P < .001]; AO, 7.2 [1.7] [not siginifcant]; RA, 11.0 [1.4] [P = .01]). Figure 4C illustrates the effect of estrogen treatment in UV-B–irradiated animals, inducing proliferation and exaggeration of all epidermal cellular layers. A peptide-treated mouse skin sample showing a near-normal reversal of the irradiation effect is shown in Figure 4D. A similar effect on epidermal width was seen after AO treatment. Figure 4E-F illustrate irradiation-induced proliferation of PCNA-positive cells in the basal layer of the epidermis following UV-B irradiation (Table 2).
Aging populations globally are concerned about clinical improvements of the face damaged by intrinsic aging and photoaging, with the inescapable alteration of appearance, and encounter a bewildering variety of products that claim to restore the aging face. Topical therapies that are proven to be fruitful clinically or experimentally can also enhance the effect of invasive measures. Cosmetic skin care has gradually unfolded as a science with inputs from basic sciences and preventive medicine. Clinicians in the field of facial plastic surgery are increasingly using these cosmetic products and nutritional supplements for amelioration of facial rhytid reduction.12 Many patients show interest in cost-effective noninvasive measures to improve skin appearance, and the practicing clinician is sought after for advice about addressing modalities of facial aging.13 Therefore, clinical research studies are being conducted to estimate the safety and benefits of topical skin care products for treating photodamaged skin.14- 18
Among the most researched products, ascorbic acid, peptides, retinoid acid derivatives, and alphahydroxy acids seem to exert promising antiaging effects.19 Indeed, under our experimental conditions among all 4 tested agents, the peptide cream and the AO mixture produced overall the best reversal of changes occurring in the photoaged hairless mouse. Application of peptide products or growth factors for wrinkle effacement and treatment of the photoaged face has recently become a popular option because these agents can influence cellular metabolic activities or act as signaling agents when used as cosmeceuticals. In the present study, the peptide cream showed its reversal effect on irradiated mouse skin in terms of all 3 parameters (ie, surface feature, epidermal thickness, and PCNA index). To our knowledge, evidence of a topical effect of peptide preparation under in vivo condition in a photoaged animal model has never been explored, although it was shown that collagen-like peptides produced antiwrinkle effects on human skin, as shown in silicone replica analysis.20 Peptides can decrease UV-B–induced erythema in human skin.21 It has been cautioned, however, that there may be adverse consequences of introducing transcription factors into cells.22 Whether peptide products are endowed with the capacity to quench the action of free radicals similar to AOs has not been illustrated, but the anti-inflammatory property of topical peptides and reversal of photodamage is notable in this study.
In this experiment, AO mixture also produced some noticeable improvement in irradiated animals (ie, surface features, epidermal thickness). The same kind of AO mixture was found to attenuate harmful UV effects in human skin.23 In the hairless mice, oral administration of an AO mixture reversed photoaging effects.24 UV radiation is a cumulative process that is initiated by the photochemical generation of reactive oxygen species ultimately causing deleterious modification of cellular machinery,25 and AOs in topical preparations can act as scavengers of free radicals generated from oxidative damage in UV-damaged akin.26
The protective effect of estrogen against photoaging in our experimental mice was not clear-cut, although some degree of reduction in Rz (roughness) values was observed. On the other hand, aggravation of irradiation-induced increase in epidermal thickness and PCNA index was noted in animals exposed to topical estrogen application. Clinical studies have shown that topical estrogen treatment in patients enhanced epidermal and dermal thickness27; however, topical estrogen treatment did not stimulate collagen production in photodamaged human skin.28
In the present experiment, a reversal of photoaging was not observed with the retinoid preparation. The effect of RA on epidermal thickness in UV-irradiated hairless mouse skin has been equivocal as reported by others. Our results agree with that of other researchers29,30 who reported that increased epidermal thickness induced by UV-B was further enhanced by long-term retinoid treatment. Topical tretinoin produced a marked increase in epidermal thickness in intrinsically aged skin of elderly women when applied for 9 months.31
Photographs of surface replicas of hairless mouse skin have been described in numerous publications32 and normally shows the abundance of lines, fine wrinkles, or buttonlike projections, which were described as bulging outlines of hair follicles.33 The wrinkle formation in photoaged hairless mice has been studied in numerous published articles, but attention was never given to these small buttonlike projections, which may represent epidermal “bubble- or global-like” structures consisting of keratinocytes or rudimentary follicles as described in scanning electron microscopic studies.34 In the present study we noted a quantitative reciprocity between the bumps and wrinkles under irradiated conditions. Whether the disappearance of bumps in irradiated skin is more apparent than actual, resulting simply from abundant coarse wrinkle formation leading to overshadowing of the bumps, has to be verified through further scanning electron microscopic studies.
Photoaging effects on hairless mouse skin have been extensively reported, and the reversal or prevention of the irradiation effect is being investigated by clinicians and scientists. An amazing variety of commercial topical antiaging or wrinkle-effacing preparations using different strategies is currently available. Laboratory or clinical experiments to test or compare the relative efficacy of such products have not been very extensively reported but can point out relevant information about commercial cosmeceuticals.35 The present study investigating some popular antiaging compounds was aimed to quantify a few epidermal morphological parameters and surface roughness to evaluate on a comparative scale, and the information will be helpful to clinicians and patients.
Accepted for Publication: November 8, 2013.
Corresponding Author: Tapan K. Bhattacharyya, PhD, Department of Otolaryngology–Head & Neck Surgery, University of Illinois at Chicago, 1855 W Taylor St, Eye and Ear Infirmary Bldg 902, Mail Code 648, Chicago, IL 60612 (firstname.lastname@example.org).
Published Online: May 29, 2014. doi:10.1001/jamafacial.2013.2582.
Author Contributions: Drs Bhattacharyya and Pathria had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Bhattacharyya, Thomas.
Acquisition, analysis, or interpretation of data: Bhattacharyya, Pathria, Mathison, Vargas.
Drafting of the manuscript: Bhattacharyya.
Critical revision of the manuscript for important intellectual content: Bhattacharyya, Pathria, Mathison, Vargas, Thomas.
Statistical analysis: Bhattacharyya, Pathria, Mathison, Vargas.
Administrative, technical, or material support: Mathison, Vargas.
Study supervision: Bhattacharyya, Thomas.
Conflict of Interest Disclosures: None reported.
Funding/Support: This research was supported by the Bernstein grant from the American Academy of Facial Plastic and Reconstructive Surgery Foundation.
Role of the Sponsor: The sponsor had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: The replica analysis was performed by David L. Miller, PhD (CuDerm Corp), as a paid service.