RESULTS

A. Histologic changes of photoaging 1. Routine histology (H&E staining)

In photoaging (B) group, both epidermal thickness and dermal thickness were increased comparing with the control (A) group (epidermal thickness; from 27.83 ± 1.93 μm to 54.89 ± 4.35 μm after photoaging, dermal thickness; from 145.76 ± 8.84 μm to 252.94 ± 19.15 μm after photoaging) (Fig. 1A, B) (p<0.05). Total dermal area was increased in photoaging group (from 9.55 x 10⁴ ± 0.90 x 10⁴ μm² to 1.33 x 10⁴ ± 2.85 x 10⁴ μm² after photoaging);

however, it was not statistically significant (Fig. 1B) (p=0.098).

A.

12 B.

Fig. 1. Increased epidermal thickness and dermal thickness after photoaging. The epidermal thickness and dermal thickness increased after photoaging (H&E, x 200, original magnification) (A). The mean epidermal thickness and dermal thickness was significantly increased after photoaging; however, the increment of total dermal area was not significant (B). (*p<0.05)

2. Masson-trichrome staining and Verhoeff’s elastic staining

In photoaging (B) group, the amount of dermal collagen fiber was decreased from 37.95 ± 28.76 % to 22.74 ± 9.90 % and the elastotic materials, one of the markers of the solar elastosis, were increased compared with the control (A) group (Fig. 2A, B).

13 A.

B.

Fig. 2. Decreased the amount of collagen fiber and increased elastotic materials in the dermis after photoaging. The amount of collagen fiber was decreased after photoaging (Masson-trichrome staining, x200, original magnification). (A). The elastotic materials were increased after photoaging, which is one of the indicator of solar elastosis (Verhoeff’s elastic staining, x400, original magnification) (B).

3. Immunohistochemical staining (1) Phospho-p44/42(Erk1/2)

Immunoreactivity of ERK was decreased from 1.63 ± 0.42 % to 0.84 ± 0.76 % in photoaging (group B), compared with control (group A), though not statistically significant

14 (Fig. 3).

(2) Phospho-SAPL/JNK, phospho-p38, TGF-β1, MMP-1, and MMP-3

The staining with above antibodies was minimal; therefore it was inappropriate to analysis with imaging analysis program.

Fig. 3. Decreased ERK expression after photoaging. The ERK expression was decreased after photoaging, though not statistically significant (x400, original magnification).

B. Chronological changes of mice skin after photorejuvenation induced by 5-ALA PDT 1. Routine histology (H&E staining)

During photoaging (group B), both epidermal thickness and dermal thickness were increased; however, in group E-F, they were significantly decreased from day 7. After day 7, both were similar to those of the control group (group A) (epidermal thickness of control vs.

photoaging vs. day 7 group; 27.83 ± 1.93 μm vs. 54.89 ± 4.35 μm vs. 32.83 ± 2.46 μm, dermal thickness of control vs. photoaging vs. day 7 group; 145.76 ± 8.84 μm vs. 252.94 ± 19.15 μm vs. 192.68 ± 17.69 μm). However, there was no consistent change chronologically.

Furthermore, total dermal area showed no significant difference during the observed time

15 period.

2. Masson-trichrome staining and Verhoeff’s elastic staining

After PDT, the amount of collagen fiber was increased through day 2 to day 21, especially, on day 21 after PDT with 40J/cm² compared with photoaging group (photoaging vs. day 21 after PDT; 22.74 ± 9.90 % vs. 50.47 ± 15.21 %) (p<0.05) (Fig. 4A). Degraded elastic fibers and increased elastotic materials during photoaging are diminished after 5-ALA PDT (Fig.

4B). The increased elastotic materials, which are known to be one of the histologic hallmarks of photoaging, were reversed by 5-ALA PDT, which means that 5-ALA PDT has an effect on photorejuvenation. With LED only group (group D), the amount of collagen fibers in the dermis was slightly increased comparing with photoaging group; however, it was not statistically significant. Among group E1, E2, and E3, intervened with different number of total sessions of PDT, the dermal collagen amount showed minimal differences.

16 A.

B.

Fig. 4. Increased the amount of collagen fiber and decreased elastotic material in the dermis after 5-ALA PDT in photoaged mice skin. The amount of collagen fiber was increased through day 2 to day 21, especially, on day 21 after PDT with 40J/cm² compared with photoaging group (p<0.05) (A). Degraded elastic fibers and increased elastotic materials during photoaging are diminished after 5-ALA PDT (B).

17 3. Immunohistochemical staining

(1) Phospho-p44/42(Erk1/2)

After PDT, the ERK expression seemed to be increased after PDT with 20J/cm² compared with phogoaging group (Fig. 5); however, it was not significant as well as not consistent result during the observed time period.

(2) Phospho-SAPL/JNK, phospho-p38, TGF-β1, MMP-1, and MMP-3

The staining with above antibodies was minimal; therefore it was inappropriate to analysis with imaging analysis program.

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Fig.5. Slightly increased ERK expression after 5-ALA PDT in photoaged mice skin. The ERK expression was increased after 5-ALA PDT in photoaged mice skin, though not statistically significant. Furthermore, there was no consistent change during the observed time period.

C. Transmission electron microscopy: changes in the quantities of collagen fibers and fibroblasts

1. Changes in collagen fibers

After photoaging, the collagen fibers (c) were much decreased in the dermis (arrow) compared with control group. However, during 5-ALA PDT application, the decreased collagen fibers were remarkably increased which was filled entire dermis. The increment was noticeable from day 2 after PDT. Furthermore, the amount of collagen fiber from day 2 after PDT exceeded that of control group (Fig. 6).

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Fig. 6. Differences in the amount of collagen fibers between photoaging, and 5-ALA PDT group with transmission electron microscopy. After photoaging, the collagen fibers were much decreased compared with control group. However, the decreased collagen fibers were remarkably increased after 5-ALA PDT application. The increment of the amount of collagen fibers was noticeable from day 2 after PDT which exceeded those of control group (arrow: dermis, (c): collagen fibers).

2. Changes in fibroblasts

Normal dermal fibroblast (f) with normal endoplasmic reticulum was observed in control

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group. During photoaging, much distended dermal fibroblast with much distended endoplasmic reticulum was noted, which means that procollagen synthesized by endoplasmic reticulum cannot be drained freely from fibroblasts. However, after PDT application, healthy fibroblast with normalized endoplasmic reticulum was observed. Also, the electrodense collagen fiber was much more attached with the surface of the fibroblast after PDT application. It seems that the fibroblast is working well with no collagen attenuation in endoplasmic reticulum (Fig. 7). Furthermore, in PDT group, the function of the fibroblast much exceeded that of control group. All above results are summarized on Table 3.

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Fig. 7. Differences of fibroblasts among control, photoaging, and 5-ALA PDT group with transmission electron microscopy. Normal dermal fibroblasts with normal endoplasmic reticulum were observed in control group. During photoaging, much distended dermal fibroblast with much distended endoplasmic reticulum was noted. However, after 5-ALA PDT application, healthy fibroblast with normalized endoplasmic reticulum was observed. It seems that the function of fibroblast with no collagen attenuation in endoplasmic reticulum also normalized after PDT application ((f): fibroblast).

22 5-ALA, 5-aminolevulinic acid; PDT, photodynamic therapy

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IV. DISCUSSION

Photoaging is initiated by cumulative process of environmental damages such as UV irradiation and involves complex alterations of the structural components of the dermal extracellular matrix, especially collagen and elastic fibers (Chung et al., 2001). The histologic hallmarks of photoaging include the loss of collagen and the deposition of elastotic material (Fisher et al., 1997; Lewis et al., 2004). Recently, the increasing interest in reversing photoaging led to emerge the various modalities in dermatologic field, such as chemical peeling and non-ablative and ablative devices.

Topical PDT was originally used for superficial nonmelanoma skin cancers and their precursors. Recently, the efficacy of PDT in other benign diseases, such as acne vulgaris, sebaceous gland hyperplasia, and hidradenitis supprativa, has been introduced (Szeimies et al., 2002). Furthermore, preventing and reversing photoaging by PDT, photorejuvenation, has been widely used (Ruiz-Rodriguez et al., 2002).

The most common photosensitizers used in dermatology are ALA and methyl amonilevulinate (MAL). ALA–PDT utilizes 20% 5-ALA as a prodrug, which, when incorporated in the skin, becomes active in the form of protoporphyrin IX (PpIX) (Gold, 2009). MAL–PDT is the methyl ester of ALA. It also is a prodrug that, when applied to the skin, is converted to PpIX. In contrast to ALA, MAL is more lipophylic, which means it can absorb more deeply into appropriate skin cells (Gold, 2009). Both of them are used in nonmelanoma skin cancer treatment with similar efficacy. There are some reasons why I have chosen ALA as photosensitizer in this study. First, there are much more clinical studies

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with ALA-PDT in photorejuvenation than those with MAL-PDT. Previous studies demonstrated that ALA-PDT treatment with blue light source had effect on the photorejuvenation and ALA application with intense pulse light (IPL) increased type I collagen production (Gold, 2002; Marmur et al., 2005). Also, the comparative split-face study demonstrated that the 5-ALA-PDT-IPL-treated sides showed greater improvement than the IPL-treated side (Kosaka et al., 2010). On the other hand, the molecular studies and studies with aminal model were little reported so far. The immunohistochemical study with ALA-PDT has been reported by our institute (Park et al., 2010) and the animal model study with MAL-PDT has been introduced recently (Choi et al., 2010). Also, the lack of mice model study of photorejuvenation with ALA-PDT led to initiate this study. Furthermore, the application of photosensitizer on the dorsal skin of mice is much easier with ALA, the liquid form, than MAL, the ointment form.

In this study, I observed the alteration of collagen and elastic fibers among control, photoaging, and post-ALA-PDT in photoaged group. There were numerous clinical observation studies with small sample sizes showing the changes of collagen and elastic fibers after PDT application (Gold, 2002; Marmur et al., 2005; Kosaka et al., 2010). With histological evaluation in animal model, I confirmed the significant decrease in collagen fibers during photoaging (the amount of dermal collagen fiber, from 37.95 ± 28.76 % to 22.74 ± 9.90 %). Also, the decreased collagen fibers by photoaging was reversed with ALA-PDT significantly (the amount of dermal collagen fiber, 45.10 ± 10.56 % at day 20 with ALA-PDT 20J/cm² and 50.47 ± 15.21 % at day 20 with PDT 40J/cm²). However, they did not showed in a specific time-dependent manner. In terms of alteration of elastic fibers, the increased

25

elastotic materials during photoaging were also reversed with ALA-PDT. However, comparing between PDT with light dose of 20J/cm² and 40J/cm², the collagen fibers seemed not to increase in a dose-dependent manner. Also, with the total two or three sessions of PDT, the amount of collagen fiber showed no significant difference comparing with one session of PDT. It is postulated that one session of PDT might be enough to induce the effect of photorejuvenation. Further studies to demonstrate adequate dose, adequate intervals between sessions, and the number of total sessions of PDT for photorejuvenation will be investigated.

Both epidermal and dermal thicknesses were increased during photoaging, representing the clinical changes of photoaged skins. After UV irradiation, the skin became thickened and rough with coarse wrinkles (Rabe et al., 2006). However, the changes were reversed with 5-ALA-PDT application, which represents that the clinical changes of photoaged skin were also reversed.

To investigate the upstream pathway of producing collagen fiber, immunohistochemical studies with antibodies to MMPs, TGF-β, and MAP kinase were performed. However, the staining with MMPs, TGF-β, JNK, and p38 showed minimal expression not enough to evaluate with imaging analysis. With monoclonal antibodies to ERK, I observed the decreased expression during photoaging. During ALA-PDT, the ERK expression seemed to be slightly increased; however, there was no certain rule of increment depending on the dose intensity, the number of sessions, or variable time period. MAP kinase is one of the signal transduction pathways associated with transcription factors, such as activating protein-1 (AP-1) (Maziere et al., 2003). Families of MAP kinases include ERK, JNK, and p38 which have numerous crosstalks among themselves (Roux and Blenis, 2004). It has been insisted that the

26

balance between the growth factor-activated ERKs and the stress-activated JNKs and p38 pathways influences on the cell survival in the stressful environment (Bickers and Athar, 2006). However, our results were not sufficient to confirm the changes of MAP kinases during photorejuvenation, because the immunohistochemical studies were not enough to investigate the transiently changing signal transduction pathways. Further investigations in molecular levels will be needed. MMPs play a role in the degradation of extracellular matrix components associated with tissue remodeling (Brennan et al., 2003). Fisher et al. (1997) showed that UV irradiation induces MMPs synthesis in human skin in vivo, following collagen degradation (Fisher et al., 1997). During PDT, the decrease in the level of MMPs could play a role in photorejuvenation (Park et al., 2010). However, in this study, the staining with antibodies to TGF-β as well as MMPs showed minimal. It seems that the skin specimens of mice are harder to study with monoclonal antibodies than those of human.

With TEM studies, I have observed the alterations of the collagen fibers and dermal fibroblasts. The degradation of electrodense collagen fibers after photoaging was consistent with the immunohistochemical studies. Furthermore, the reversal of collagen amount after ALA-PDT was also confirmed with TEM. Especially, the increment of collagen fiber was noticeable from day 2 after ALA-PDT, which was prior to the change in immunohistochemical studies. The previous studies showed that the proinflammatory cytokines induced after PDT treatment led to the dermal remodeling, such as degradation of fragmented collagen in early phase (Choi et al., 2010). With TEM, I demonstrated that the collagen fiber started increasing even during early inflammatory phase right after ALA-PDT.

The fibroblast normally has extensive rough endoplasmic reticulum which is actively

27

synthesizing protein for export (Breathnach, 1971). When the fibroblast cannot export collagen fiber to the dermis enough, the endoplasmic reticulum would be extended with the procollagen materials (Breathnach, 1971). These changes of fibroblast were observed in photoaged group; however the healthy and well-functioning fibroblast with normalized endoplasmic reticulum reappeared after ALA-PDT application.

There were several previous studies with TEM in photoaged model. Inomata et al. observed the destruction of basement membrane structure and dermal collagen fibers in chronically UV-irradiated mouse (Inomata et al., 2003) and Watson et al. demonstrated the increased dystrophic elastic fibers with sparse microfibrillar apparatus after photoaging and also demonstrated reversely increased deoposition of the microfibrillar dermal matrix components after 4-hr occlusive 0.025% retinoic acid application (Watson et al., 2001). They were focused on the dermal matrix; however, there was no report with the observation of fibroblast after photoaging or phogorejuvenated tools. It is remarkable point that the ALA-PDT could affect normalizing the function of fibroblast itself in this study, which had been more inactive during photoaging, as well as the increment of collagen fibers. It represents that the ALA-PDT application induces not only the transient increment of the amount of collagen fibers but also long-term effect with normalizing function of fibroblast.

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V. CONCLUSION

In conclusion, this study provided the histologic evidence of the beneficial effects of ALA-PDT for photodamaged skin in mice model. ALA-ALA-PDT induces the deposition of collagen fibers in the dermis and normalizes the dystrophic elastotic materials induced by photoaging.

However, the change did not correlate with the course of time, the light dose of PDT, or the total number of sessions of PDT. With TEM, ALA-PDT might have a direct influence on the normalizing function of fibroblast as well as the increment of collagen fibers. Further studies will be necessary to fully determine the adequate parameters of PDT for photorejuvenation.

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REFERENCES

1. Bickers DR, Athar M: Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol 126: 2565-2575, 2006

2. Breathnach AS: An atlas of the ultrastructure of human skin: development, differentiation, and post-natal features, Churchill, 1971

3. Brennan M, Bhatti H, Nerusu KC, Bhagavathula N, Kang S, Fisher GJ, Varani J, Voorhees JJ: Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol 78: 43-48, 2003

4. Chen A, Davis BH: UV irradiation activates JNK and increases alphaI(I) collagen gene expression in rat hepatic stellate cells. J Biol Chem 274: 158-164, 1999

5. Choi JY, Park GT, Na EY, Wi HS, Lee SC, Lee JB: Molecular changes following topical photodynamic therapy using methyl aminolaevulinate in mouse skin. J Dermatol Sci 58: 198-203, 2010

6. Chung JH, Seo JY, Choi HR, Lee MK, Youn CS, Rhie G, Cho KH, Kim KH, Park KC, Eun HC: Modulation of skin collagen metabolism in aged and photoaged human skin in vivo. J Invest Dermatol 117: 1218-1224, 2001

7. Davis BH, Chen A, Beno DW: Raf and mitogen-activated protein kinase regulate stellate cell collagen gene expression. J Biol Chem 271: 11039-11042, 1996

8. Fisher GJ, Datta S, Wang Z, Li XY, Quan T, Chung JH, Kang S, Voorhees JJ: c-Jun-dependent inhibition of cutaneous procollagen transcription following ultraviolet

30

irradiation is reversed by all-trans retinoic acid. J Clin Invest 106: 663-670, 2000 9. Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ: Pathophysiology of

premature skin aging induced by ultraviolet light. N Engl J Med 337: 1419-1428, 1997

10. Gold MH: The evolving role of aminolevulinic acid hydrochloride with photodynamic therapy in photoaging. Cutis 69: 8-13, 2002

11. Gold MH: Therapeutic and aesthetic uses of photodynamic therapy part five of a five-part series: ALA-PDT and MAL-PDT what makes them different. J Clin Aesthet Dermatol 2: 44-47, 2009

12. Gold MH, Bradshaw VL, Boring MM, Bridges TM, Biron JA: Split-face comparison of photodynamic therapy with 5-aminolevulinic acid and intense pulsed light versus intense pulsed light alone for photodamage. Dermatol Surg 32: 795-803, 2006 13. Gum R, Wang H, Lengyel E, Juarez J, Boyd D: Regulation of 92 kDa type IV

collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene 14: 1481-1493, 1997 14. Inagaki Y, Truter S, Ramirez F: Transforming growth factor-beta stimulates alpha

2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem 269: 14828-14834, 1994

15. Inomata S, Matsunaga Y, Amano S, Takada K, Kobayashi K, Tsunenaga M, Nishiyama T, Kohno Y, Fukuda M: Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse. J Invest Dermatol 120: 128-134, 2003

31

16. Kahari VM, Saarialho-Kere U: Matrix metalloproteinases in skin. Exp Dermatol 6:

199-213, 1997

17. Kosaka S, Yasumoto M, Akilov OE, Hasan T, Kawana S: Comparative split-face study of 5-aminolevulinic acid photodynamic therapy with intense pulsed light for photorejuvenation of Asian skin. J Dermatol 37: 1005-1010, 2010

18. Lewis KG, Bercovitch L, Dill SW, Robinson-Bostom L: Acquired disorders of photorejuvenation: a pilot study. J Cosmet Laser Ther 7: 21-24, 2005

21. Maziere C, Floret S, Santus R, Morliere P, Marcheux V, Maziere JC: Impairment of the EGF signaling pathway by the oxidative stress generated with UVA. Free Radic Biol Med 34: 629-636, 2003

22. Montagna W, Kirchner S, Carlisle K: Histology of sun-damaged human skin. J Am Acad Dermatol 21: 907-918, 1989

23. Park MY, Sohn S, Lee ES, Kim YC: Photorejuvenation induced by 5-aminolevulinic acid photodynamic therapy in patients with actinic keratosis: a histologic analysis. J Am Acad Dermatol 62: 85-95, 2010

24. Quan T, He T, Kang S, Voorhees JJ, Fisher GJ: Solar ultraviolet irradiation reduces

32

collagen in photoaged human skin by blocking transforming growth factor-beta type II receptor/Smad signaling. Am J Pathol 165: 741-751, 2004

25. Rabe JH, Mamelak AJ, McElgunn PJ, Morison WL, Sauder DN: Photoaging:

mechanisms and repair. J Am Acad Dermatol 55: 1-19, 2006

26. Roux PP, Blenis J: ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68: 320-344, 2004

27. Ruiz-Rodriguez R, Sanz-Sanchez T, Cordoba S: Photodynamic photorejuvenation.

Dermatol Surg 28: 742-744, 2002

28. Shin MH, Rhie GE, Kim YK, Park CH, Cho KH, Kim KH, Eun HC, Chung JH:

H2O2 accumulation by catalase reduction changes MAP kinase signaling in aged human skin in vivo. J Invest Dermatol 125: 221-229, 2005

29. Szeimies RM, Landthaler M, Karrer S: Non-oncologic indications for ALA-PDT. J Dermatolog Treat 13 Suppl 1: S13-18, 2002

30. Touma D, Yaar M, Whitehead S, Konnikov N, Gilchrest BA: A trial of short incubation, broad-area photodynamic therapy for facial actinic keratoses and diffuse photodamage. Arch Dermatol 140: 33-40, 2004

31. Watson RE, Craven NM, Kang S, Jones CJ, Kielty CM, Griffiths CE: A short-term screening protocol, using fibrillin-1 as a reporter molecule, for photoaging repair agents. J Invest Dermatol 116: 672-678, 2001

32. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME: Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326-1331, 1995

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