Results

The Dermainspect was utilized to perform two studies in the skin of volunteers. In both studies the morphology of the mitochondrial network in epidermal keratinocytes was investigated based on the autofluorescence of NADH. All volunteers provided healthy skin of either skin type II or skin type III of Fitzpatrick’s phototyping scale. [102] Two weeks before and during the studies volunteers desisted visits of solariums and inten-sive sun exposure. Furthermore, treatment of both, medical substances and cosmetics, was prohibited during the studies. All volunteers were non-smokers. Both studies were performed in accordance with the recommendations of the current version of the Dec-laration of Helsinki [103] and the guideline of the International Conference on Harmo-nization Good Clinical Practice (ICH GCP). [104] Prior the examinations volunteers were provided with fifteen minutes of acclimation to the measurements under standard atmospheric conditions with 23^◦C±1^◦C and 43%±2% relative humidity level. All measurements were carried out by trained personnel.

Examinations were performed at the inner side of both forearms of each volunteer. For optical immersion a drop of water was positioned at the investigated spot of the skin.

Parallel, by using a magnetic adapter, a thin platelet of glass was placed under the objective with immersion oil between the lens and the glass (Figure 4.7). Following these preparations, the objective approached the skin until the focus reached epidermal and dermal layers. All volunteers in both studies were investigated at two areas of each forearm. Measurements at three different positions in each area were acquired. All mea-surements had an acquisition time of about 12.5 s and dimensions of 110µm ×110µm

objective

arm

water adapter oil

glue

glass

A drop of water is placed on the inner side of the forearm. Immersion oil is placed between the objective and a glass platelet. The three elements are hold together by an magnetic adapter. Then, the objective approaches the arm until the focus of the laser reaches epidermal layers. To prevent movements of the investigated spot relatively to the objective, the skin sticks to the adapter by adhesive tape.

Statistical analysis of morphological parameters was conducted with the software Sta-tistica by StatSoft (Europe) GmbH (Hamburg, Germany). [105] The unpaired samples in the aging study were compared by a Mann-Whitney U test, the paired samples in the differentiation study by a Wilcoxen test. [106] As the p-value for significance the thresholdp=0.05 was chosen.

In the following subsections the basic results of both studies are presented. Further analysis and discussion are presented in the publication at the end of the chapter.

4.2.1 Aging of Skin

In this study, the morphology of the mitochondrial network in the skin of young and old volunteers was compared. For that purpose, the autofluorescence of keratinocytes in the stratum granulosum was imaged. The study consists of measurements in the epidermis of twelve young (mean ±SD: 23.75±1.67 years) and twelve old (mean ±SD: 72.17± 4.15 years) volunteers.

After experimental examinations and binarization, quantitative analysis of

mitochon-chondrial clusters. Large mitomitochon-chondrial clusters are defined as connected fluorescing areas that are larger than the average cluster size in measurements of young volunteers Ayoung = 9.05 pixels. Hence, large clusters consist of ten or more pixels. Before statisti-cal comparison between both age groups, the results in each area at the forearms were averaged.

Results for all parameters are presented in Figure 4.8. Analysis disclose that the total amount of autofluorescence of NADH in keratinocytes of cells is significantly higher in old volunteers than in young volunteers (p≤0.001). Additionally, micrographs show, that cross-sections of single keratinocytes in old volunteers cover greater areas than keratinocytes of young volunteers. [107] Thus, for a relative comparison, the total amount of autofluorescence has to be normalized to the area of the cytoplasm of the cell. Then, a comparison do not reveal any significant differences between young and old skin (p=0.826).

Quantitative analysis of the morphological parameters of all mitochondrial clusters re-veals, that the mitochondrial network is more fragmented in old skin than in young skin.

There is a significantly higher number n of mitochondrial clusters in keratinocytes of old volunteers than of young volunteers (p≤0.001). Furthermore, clusters in cells of old skin establish a significantly lower circularity C (p=0.015) and a significantly smaller cluster size A (p=0.035). Hence, mitochondrial clusters in cells of old volunteers are more compact and smaller than in cells of young volunteers.

A quantitative analysis of the morphology of large mitochondrial clusters discloses a significantly higher number of large mitochondrial clusters n_large (p=0.039) in old skin.

Additionally, large mitochondrial clusters exhibit higher values for the large cluster size Alarge (p=0.084) and have a significantly higher circularityClarge (p=0.002) in old skin.

Thus, large mitochondrial clusters have a tendency to be more complex in the stratum granulosum of old skin.

4.2.2 Epidermal Differentiation

This study was performed in order to investigate alterations in the morphology of the mitochondrial network during the differentiation of keratinocytes in the epidermis. For

a) b)

c) d)

e) f)

g) h)

Investigated parameters of the aging study are compared between young and old volunteers by statistical boxplots. The p-value represents the probability if the two compared probability distributions derive from the same population. The square in the box represents the mean value, the horizontal line in the center the median. 25%

percent of the measured values are lower than the bottom of the box, 75% of them are lower than the top of the box. The antennas (whiskers) are the corresponding limits for

after annotation and binarization procedures the three morphological parameters n, A and C were investigated for all mitochondrial clusters per cell and for large mitochon-drial clusters.

Results for all parameters are presented in Figure 4.9.

As cross-sections of keratinocytes in the stratum spinosum are smaller than in the stra-tum granulosum, there is less autofluorescence in total in these cells. However, normaliz-ing the amount of autofluorescence to the cell’s area discloses a higher density of NADH related signal in the stratum spinosum than in the stratum granulosum.

Quantitative analysis for all clusters reveals a significantly higher number n of mi-tochondrial clusters in keratinocytes in the stratum granulosum than in the stratum spinosum (p≤0.001). Moreover, mitochondrial clusters in the stratum spinosum have a significantly higher average size A (p≤0.001) and a significantly higher circularity C (p≤0.001). Hence, mitochondrial clusters in less differentiated keratinocytes in the stra-tum spinosum establish larger, more complex structures than the more differentiated cells in the stratum granulosum. Thus, during differentiation of keratinocytes the mito-chondrial network performs a fragmentation process. Differences between mitomito-chondrial structures of different epidermal layers are similar to the ones of different age groups.

Investigating the same morphological parameters for large mitochondrial clusters reveals a significantly greater average sizeAlarge (p≤0.001) and a significantly higher circular-ity C_large (p≤0.001) in the stratum spinosum. These findings support the notion of a fragmented mitochondrial network in keratinocytes at the end of their differentiation process in the stratum granulosum. However, analysis of large mitochondrial clusters also discloses a significantly higher number of mitochondrial clustersn_large(p≤0.001) in the stratum granulosum.