ANIMALS AND MICE GROUPING - Histological Changes in Photorejuvenation Induced by 5-Aminolevulin

6-week-old female albino hairless mice (Skh:hr-1) weighed 25–30 g were obtained from Oriental Co., Ltd. and maintained under individually ventilated cage (IVC) system. All experimental protocols were approved by the Committee for Animal Care and Use at Ajou University. Throughout the experimental protocol, the mice were maintained at standard conditions - temperature 24 ± 2℃, relative humidity 50 ± 10%, and 12 h room light ⁄ 12 h dark cycle. Mice were divided into six groups: control group (group A), photoaging group (group B), ALA only group (group C), light-emitting diode (LED) only group (group D), 5-ALA PDT group with 20J/cm² of light dose (group E), and 5-ALA PDT group with 40J/cm² of light dose (group F). For group E, it was divided to 3 identical groups; which were performed from 1 session to 3 sessions of 5-ALA PDT to investigate the differences among the number of total sessions of PDT (Table 1).

4 Table 1. Mice grouping

Groups Methods No. of mice

A Healthy control* 4

B Photoaging 4

C 5-ALA only 4

D LED only (20J/cm²) 4

E 5-ALA PDT (20 J/cm²) 12 ^†

E1 5-ALA PDT (20 J/cm²) x 1 session 4 E2 5-ALA PDT (20 J/cm²) x 2 sessions 4 E3 5-ALA PDT (20 J/cm²) x 3 sessions 4

F 5-ALA PDT (40 J/cm²) 4

5-ALA, 5-aminolevulinic acid; LED, light-emitting diode; PDT, photodynamic therapy

* Neither photoaging nor PDT

† There were 3 identical groups; which were performed from 1 session to 3 sessions of ALA PDT to

investigate the differences among the number of total sessions.

5 B. UV IRRADIATION FOR PHOTOAGING

For group B to F, UV irradiation was obtained using TL 20W/12 RS UV lamps (Philips, Eindhoven, The Netherlands, range 275-380 nm, peak 310-315 nm). A Kodacel filter (TA401/407; Kodak, Rochester, NY) was mounted 2 cm in front of the UV lamps to remove UVC wavelengths < 290 nm. The UV-dose was measured in minimal erythema dose (MED) using a UV meter (Model 585100; Waldmann Co., Villingen-Schwenningen, Germany).

MED is defined as the minimum amount of radiation exposure required to produce erythema with sharp margins after 24h and MED was 100 mJ/cm². During 5 weeks, the UV irradiation was administrated from the initial dose of 1.3 MED (130 mJ/cm²) to 3 MED (300 mJ/cm²) three times weekly. The UV dose was increased weekly by 0.5 to 1 MED up to 3 MED, listed on Table 2. The administrated distance from dorsal skin surface of mice was approximately 17.5 cm.

Table 2. UV irradiated dose for each week and the cumulative irradiated dose

Week Sessions Dose (mJ/cm²)

7 C. PDT FOR PHOTOREJUVENATION

I used 20% 5-ALA (Levulan®, Dusa Pharmaceuticals, Wilmington, MA) as photosensitizer.

The solution was applied on the dorsal skin of the mice and the animals were kept in a dark room for 4 hour and the remaining solution was wiped by ethanol and normal saline. The photosensitizer was applied on group C, E, and F.

The LED light irradiation was performed on group D, E, and F, using an incoherent light source (Omnilux reviveTM, 633nm, Phototherapeutics Ltd., Montgomeryville, PA, USA).

D. SERIAL SKIN BIOPSY

After 3 days of resting from the irradiation, all mice got inhalation anesthesia, and serial skin biopsies were performed from each mouse at day 2, day 7, day 14, and day 21 from each intervention. For group E, the additional skin biopsies were performed at day 28 from each session.

E. HISTOLOGIC EXAMINATION

Hematoxylin & eosin staining (H&E), Masson-trichrome staining, and Verhoeff’s elastic staining were performed. Also immunohistochemical staining was performed.

1. H&E staining

Skin biopsies were fixed in 10% formalin, embedded in paraffin, and sectioned into 5-µm sections for routine H&E staining. The epidermal thickness, dermal thickness, and total dermal area were measured. The epidermal thickness was the mean length between the outer sides of the epidermis, excluding the stratum corneum, and the dermo-epidermal

junction through the entire section. The dermal thickness was defined as the mean length between the dermo-epidermal junction and upper margin of subcutaneous fat layer. Total dermal area was calculated by the total area from the dermo-epidermal junction to upper margin of subcutaneous fat layer, excluding adnexal structure. Computer-based software (Image-Pro Plus, Media Cyberneticsa, Silver Spring, MD, USA) was used for each measurement. All slides were evaluated under constant magnification (x200) by two observers.

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

Masson-trichrome (with light green rather than aniline blue) stained collagen fibers and Verhoeff’s stained elastic fibers were quantified using software as mentioned above. For the scanning view of the papillary and upper reticular dermis, constant magnification (x200) was used. In Masson-trichrome stained slides, the amount of dermal collagen fiber was calculated as the ratio of stained area to measured area (SA/MA), presented as the percent expression. The degree of solar elastosis was evaluated as the amount of dystronic or fragmented elastic fiber in the Verhoeff’s stained slides under constant magnification (x400).

3. Immunohistochemical staining

Using the immunoperoxidase technique, 5-µm sections from formalin-fixed, embedded skin samples were stained with antibodies as follows: Rabbit monoclonal phospho-p44/42 MAPK (Erk1/2) antibody (diluted 1:100, Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal phospho-SAPL/JNK antibody (diluted 1:100, Cell Signaling Technology), rabbit monoclonal phospho-p38 MAPK antibody (diluted 1:50, Cell Signaling Technology),

rabbit polyclonal MMP-1 antibody (diluted 1:100, Abnova, Walnut, CA, USA), rabbit monoclonal MMP-3 antibody (diluted 1:100, Epitomics, Burlingame, CA, USA), and rabbit monoclonal TGF-β1 antibody (diluted 1:100, Bioworld Technology, Newmarket Suffolk, UK).

For quantitative evaluation of the immunoreactivity of ERK, JNK, p38, MMP-1, MMP-3, and TGF- β1, the previously mentioned software was used for the immunohistochemically stained sections in the same way it was used for collagen and elastic fiber measurements as percent expression.

F. TRANSMISSION ELECTRON MICROSCOPY

For transmission electron microscopic evaluation, mice skin specimens from each group were used. The specimens were fixed with Karnovsky’s fixative solution (1%

paraformaldehyde, 2% glutaraldehyde, 2 mM calcium chloride, 100 mM cacodylate buffer, pH 7.4) for 2 h and washed with cacodylate buffer. After post-fixing with the fixative solution containing 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h, tissues were stained with en bloc in 0.5% uranyl acetate, embedded in Poly/Bed 812 resin (Pelco, Redding, CA, USA). The tissues were sectioned by Reichert Jung Ultracut S (Leica, Wetzetlar, Germany) and stained with uranyl acetate and lead citrate. These specimens were observed and photographed under transmission electron microscope (Zeiss, Leo, Oberkochen, Germany).

10 G. STATISTICAL ANALYSIS

A Student’s t test and one-way ANOVA test were used to analyze the statistically significant differences among the staining patterns of biopsy specimen using the SPSS 11.0 statistics program (SPSS, Inc., Chicago, IL, USA). One-way ANOVA was initially performed to determine whether an overall statistically significant change existed before using the two-tailed Student’s t test. A value of p < 0.05 was considered statistically significant. Summary data are expressed as the mean ± standard deviation (SD).

III. 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).

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.

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.

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.

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).

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

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.

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

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

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

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

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

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

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