Martin Kaatz, MD, Department of Dermatology, Friedrich-Schiller-University, 07743 Jena, Germany, Tel.: +493641937302, Fax: +4936419374574, e-mail: email@example.com
Background: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis.
Objectives: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing.
Methods: In vivo MLT was used to image two-photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age-dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated.
Results: The resulting MLT-based Dermis Morphology Score is correlated with age (R2 = −0.90) and with the previously published SHG to AF Ageing Index of Dermis (R2 = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P < 0.001).
Conclusions: We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi-quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects.
Major features of aged skin are dryness, flaccidness, wrinkling, clinical signs of chronic UV damage, increased carcinogenesis and loss of function. Skin ageing is caused by intrinsic and extrinsic factors. Intrinsic skin ageing describes the irreversible physiological process which starts as soon as physiological maturation is accomplished. It is characterized by increasing loss of cell number, physiological morphology and function (1). Extrinsic skin ageing is mainly a consequence of cumulative UV exposure of the skin, but can be accelerated by nicotine abuse and environmental hazardous compounds. They cause specific alterations like elastosis and dyschromatic pigment shifts (2–4).
An important pathophysiological parameter for the visible process of skin ageing is the dermal collagen content. Collagen I is the major constituent of dermal extracellular matrix and its relative content decreases over age based on diminished collagen synthesis (5). The UV-induced expression of matrix metalloproteases (MMP) is an additional reason for a decreased collagen content (6). MMP activity also seems to be induced by nicotine abuse (7). The second important hallmark of skin ageing is the so called elastosis, which seems to be mainly a result from de novo synthesis of elastotic material, consisting of elastin, fibrillin and glycosaminoglycans (8–11). Extensive destruction or significant alteration of the elastic fibre network caused by direct impact of UV and MMP upregulation (12–14) might also be involved. MMP inhibitors have shown protective effects against photoageing for the basement membrane damaged by chronic UVB exposure (15,16).
Oxidative stress seems to be an important mechanism of skin ageing (17,18), and numerous approaches aim to inhibit the involved pathways, e.g. by green tea polyphenols, creatine or vitamins (19–21). The direct stimulation of collagen synthesis is object to research, e.g. by hormones like dehydroepiandrostenone and 17beta-oestradiol, or vitamins like retinoic acid derivates (22–24). Newly synthesized elastic fibres may be protected from degradation by ellagic and tannic acids (25).
So far, dermal matrix morphology has only been evaluated by histological or ultrastructural examination of previously excised skin specimens, or by in vivo methods with a low lateral resolution such as ultrasound or optical resonance tomography (26,27). MMP inhibitors have shown protective effects against photoageing for the basement membrane examined by skin fluorescence (28), but using this technique only epidermal changes may be depicted without spatial resolution. In contrast, another technique with a very high spatial resolution is atomic force microscopy that also proved to show differences between old and young skin (29). However, only the uppermost epidermal layer is accessible by this tool. Multiphoton laser imaging is suited for the non-invasive evaluation of cellular and molecular structures. In the context of skin research it has been used for imaging of dermal penetration of nanoparticle-borne drugs (30) and diffusion of other model drugs (31), powdered immunization via Langerhans cells (32), collagen dissociation by chemical (33) and physical (34) influences as well as the evaluation of skin aging by photon counting techniques (35). The use of filter systems for the separation of spectral components due to elastin and collagen has been described for the imaging of coronary artery microstructure (36). Based on total photon numbers because of autofluorescence (AF) and second harmonics generation (SHG) in optical slices of the superficial dermis, the SHG to AF Ageing Index of Dermis (SAAID) has been shown to correlate with the numerical age of humans in vivo (35).
This study was intended to demonstrate that morphological features of the dermal fibre network vary with age. These features have been defined, evaluated and weighted to calculate a score.
Materials and methods
Eighteen adult Caucasian volunteers (7 female, 11 male) aged 21–84 years [mean age (MA) 53.3] were included with informed consent. The study has been performed between January and March 2006 after approval of the local ethics committee. We included only volunteers with no dermatosis in the region of interest.
The multiphoton tomograph DermaInspect (JenLab GmbH, Jena, Germany) has been used and described previously (37). In brief, the laser device consists of a femtosecond tunable (750–850 nm) titanium sapphire laser, a galvo and piezo scanning system, a photomultiplier tube detection module for time-correlated single photon counting and a control module with image-processing hardware and software.
Multiphoton laser scanning tomography (MLT) imaging has been performed at the inner forearm about 15 cm proximal of the wrist, because of its relatively sun-protected localization, easy accessibility, little susceptibility to movement artifacts and low hair density. Five randomly chosen regions in the upper dermis were measured twice at an excitation wavelength of 820 nm and with a laser power of 49 mW. SHG images have been obtained using a 410 ± 5-nm bandpass filter and AF images with a 470-nm long pass filter. Typically, an area of 128 × 128 pixels corresponding to about 0.2 × 0.2 mm2 was imaged.
Greyscale bitmaps of all images were exported from spc-image software (Becker & Hickl GmbH, Berlin, Germany) and were subjected to blinded evaluation. Test statistics were performed with spss® 13.0 (LEAD Technologies Haddonfield, New Jersey, USA). The significance level was set to α = 0.05 and adjusted for multiple testing (αadjusted = 0.00625).
Images of the dermis were collected at five different locations of the inner forearm using multiphoton tomography. The MLT-based Dermis Morphology Score (MDMS) was developed in three steps: first, identification of morphological characteristics and their assignment to parameters and categories. Secondly, determination of the weighting of each category depending on the category MA. Thirdly, calculation of the MDMS for each subject.
All images (n = 180, 18 patients × 5 locations × 2 emission wavelength ranges) were evaluated for fibre spread and fibre aspect. Additionally, network pattern and image homogeneity were evaluated in SHG images, and clot formation in AF images. Thus, for each measurement site we evaluated a total of eight parameters (Fig.1, Table 1). For each parameter, all images were assigned to one category (e.g. straight or curled). When the image presented characteristics of both categories or none of them, the image was classified as ‘equivocal’. Out of 720 possible ratings, 435 (60.4%) have been attributed to one of the two opposed categories, while 285 (39.6%) were considered equivocal. Mann–Whitney U-tests were performed to test for age differences between the two opposed categories. For seven of the eight parameters significant differences were observed (see Table 1), and the MA of test persons whose images were evaluated as ‘equivocal’ was in between the MA of the two other groups.
Table 1. Evaluation parameters and their assigned categories
n, number of images assigned to the corresponding category; MA, SD, mean age and standard deviation of the subjects whose images were assigned to the corresponding category; Δ, difference between the overall mean age (53.3) and MA; P, P-value for non-parametric testing for age differences between the two opposed categories. The adjusted significance level is at αadjusted = 0.00625.
In the second step, we summarized the information conveyed by each of the eight parameters into a single score. As the observed morphological changes are due to degenerative processes, a plausible score had to decrease with age. The score was derived in the following way: (i) the MA of patients whose images were assigned to that category was calculated for each parameter category; (ii) for every category, an integer value Δ was calculated, resulting from the difference between that category MA and the overall MA of our sample (53.3 years). The formal equation was as follows: Δ = MA−53.3.
Finally, the score was calculated for each subject by summing their Δ values for all parameters and locations. The main results of this scoring are depicted in Fig. 2, in which each subject’s score is plotted against its corresponding numerical age. A linear regression line was fitted to describe the data (R2 = 0.90). Multiple regression of the subject’s age with the scores of the single parameters resulted in R2 = 0.97. Non-parametric testing for MDMS differences between the seven youngest and seven oldest subjects was highly significant (P < 0.001).
Recently, we described the in vivo assessment of skin ageing by another index, the SAAID (35). The results of either evaluation system are correlated (R2 = 0.66). But, in contrast to the SAAID, the MDMS failed to show gender-specific differences.
Several pathological skin conditions such as elastosis cutis, scleroderma or chronic sclerodermic graft versus host disease (GvHD) involve changes of the dermal fibre network (38–40). To distinguish those changes from physiological processes non-invasively by optical diagnostic tools, normal conditions must be evaluated first. Ageing has strong influence on the composition and morphology of the dermal matrix (41). This study shows that morphological changes of ageing skin in human dermis can be assessed by in vivo MLT.
Second harmonics generation images were used to define criteria for the description of collagen fibre morphology and evaluated for their characteristics in different age groups. Collagen fibres were judged filiform in elder subjects, while the young frequently presented an amorphous pattern in SHG images. As collagen is the major dermal matrix component in young skin, its amorphous appearance in the young is most probably due to the compact and dense arrangement of the collagen fibre bundles. The filiform arrangement in the elder is a sign for the thinned matrix because of collagen decrease of ageing skin.
As in young skin the SHG image was frequently described as amorphous, in these cases no fibres were identifiable and their spread has been judged equivocal. However, the spread of collagen fibres was described as curly in aged and straight in moderately aged subjects. This can be explained by the irregular displacement of the thinned collagen fibre network by increasing deposit of elastotic material in ageing skin. Furthermore, the decrease of dermal collagen content with age leads to the impression of a coarse fibre network and inhomogeneous images.
Autofluorescence images were used to evaluate matrix compounds other than collagen, i.e. mainly elastic fibres and elastotic material. In images of young skin, a distinct network of fine fibres was observed, while old skin showed deposits of amorphous material. The fibre spread was described as straight in young and curly in elder skin. These findings are consistent with histological grading of elastosis (42).
The origin of fluorescent clots in AF images, particularly of the young, has not clearly been elucidated. As the clots’ fluorescence intensity is higher at shorter wavelengths (data not shown), a cellular origin is probable. They may represent capillaries or stroma fibroblasts, as both decrease with age (41,43). But we were not able to clearly identify cellular structures, because of the generally low photon counts in a vertical depth of about 100 μm. Another possible origin of the fluorescent clots may be melanin pigment or melanophages. In confocal microscopy melanin has been shown to provide strong contrast (44).
Further studies with a large number of healthy volunteers will be performed to validate the evaluation parameters and to adjust the MDMS scoring points (Δ values).
Our findings on the in vivo evaluation and quantification of morphological properties of the dermal fibre network by MLT are useful for the assessment of the progress of sclerosing dermatological disorders, such as scleroderma or chronic sclerodermic GvHD. Other future applications may be the assessment of wound healing or of matrix destruction by invasive tumors.