Fractional erbium:yttrium aluminum garnet laser in the treatment of morphea mouse model

To assess the efficiency and the mechanism of fractional erbium:yttrium aluminum garnet (Er:YAG) laser for the treatment of morphea in mouse model.


| BACKG ROU N D
Morphea is an autoimmune disease characterized by skin fibrosis. 1e typical lesions of morphea are yellow-white or ivory plaques, with sclerotic in the center. 2Although the main pathophysiological change of morphea is excessive collagen synthesis and deposition, 3 the exact etiology of morphea remain unclear.Transforming growth factorβ (TGFβ) is the key profibrotic cytokine, which promotes the transcription of multiple profibrotic molecules, such as Collagen I and tissue inhibitor of matrix metalloproteinases (TIMP), leading to myofibroblast activation and matrix deposition. 4,5Matrix metalloproteinases (MMPs) is a protease family to mediate collagen cleavage and degradation of collagen.The inhibition of MMPs results in the development of fibrosis. 6TGFβ also inhibits the MMPs synthesis 7 and lead to the progress of fibrosis in morphea.Due to no effective treatment, morphea worsens patient's quality of life and even severe disability.Therefore, it is urgent to find an effective approach for the cure of morphea.
The fractional lasers form the skin microthermal treatment zones (MTZs) through photo-thermolysis, and the fractional surrounded undamaged tissue allow the MTZs rapid repair. 80][11][12] Of note, fractional carbon dioxide (CO 2 ) laser treatment could improve collagen deposition in morphea, which was superior to previous low-dose ultraviolet radiation A-1 (UVA-1) phototherapy. 13A case report revealed potential fractional erbium:yttriumaluminum-garnet (Er:YAG) laser conducive to the improvement of morphea. 14Compared with fractional CO 2 laser, fractional Er:YAG laser delivers a powerful wavelength of 2940 nm that closely approximates the absorption of light peak of water.As the skin consists of 60%-80% water, fractional Er:YAG laser results in effective ablation of target area with minimal tissue ablation and thermal damage to its surrounding tissue, producing less erythema, and adverse reaction. 15eomycin-induced dermal fibrosis is a model of the early and inflammatory stages of morphea, which have become a cornerstone of experimental biology that is highly relevant to studies of morphea. 16sed on aforementioned finds, we hypothesize that fractional Er:YAG laser could be an effective treatment of morphea and establish the bleomycin-induced morphea mouse model to evaluate the efficacy of fractional Er:YAG laser treatment based on the clinical, ultrasonic, and histopathologic assessment.

| Animal
Seven to eight weeks old male C 3 H/HeN mice, were purchased from Beijing Vital River Laboratory Animal Technology Company (License: SCXK [Jing] 2016-0011) and housed in an SPF animal laboratory with individual ventilated cages, with strictly autoclaved feed and drinking water.

| BLM-induced mouse model of morphea
Both sides of their backs were shaved before the experiment.Bleomycin (BLM) was dissolved in phosphate buffer saline (PBS) to a final concentration of 1.0 mg/mL.BLM (100 μL; BLM group; n = 15)   or PBS (100 μL; PBS group; n = 9) was subcutaneously injected into both backside of the mice daily for 21 days, using 27 G sterile hypodermic needles. 16

| Treatment interventions
In this self-controlled study, both BLM and PBS group were treated with fractional Er:YAG laser.The left side back of mouse had no treatment and set as blank control.The right back side of mice were given laser treatment once a week for 4 weeks.Treatment parameters were 100 J/cm 2 energy, 400 μm in depth, 30 Hz frequency, 2 mm aperture.

| Clinical evaluation
Clinical evaluation was performed 2 weeks posttreatment.We recorded and scored the thickness, dermal atrophy, dyschromia, and erythema according to the adjusted Localized morphea Cutaneous Assessment Tool (LoSCAT). 17

| Ultrasound evaluation
Ultrasonic image and dermal thickness were measured by highfrequency ultrasound (50 MHz) with FUJIFILM VisualSonics ultrasound device.

| Histopathologic evaluation
All mouse were sacrificed by cervical vertebra dislocation after clinical and ultrasound evaluation.Biopsies were obtained from both sides of the back of each mouse and stained with hematoxylin and eosin (H&E) for histopathologic evaluation.According to the scoring guidelines presented by Verrecchia, the degree of collagen homogeneity in papillary dermis, superficial reticular dermis, and deep reticular dermis was assessed semi-quantitatively. 18

| Collagen deposition scoring system
According to the collagen deposition scoring system, the extent of fibrous thickening was assessed semi-quantitatively as follows:

| Statistical methods
The data were statistically described as mean ± standard deviation (±SD).side of the back) showed thinned dermis, which exhibited a clear demarcation with the subcutaneous fat layer (Figure 2A).The dermis thickness was considerably higher in the untreated lesions of the BLM group than that associated with the PBS groups (p < 0.0001; Figure 2B), demonstrating the successful establishment of a morphea mouse model.The difference in dermis thickness between both sides of the back in the PBS group was not statistically significant (p > 0.05; Figure 2C).Compared with the untreated lesions, the treated lesions had lower dermis thickness in the BLM group (p < 0.001; Figure 2D), indicating that fractional Er:YAG laser treatment improved the dermal thickening of morphea lesions.

| Fractional Er:YAG laser treatment improved the pathological manifestations of mouse morphea model
The findings above promoted us to determine the histopathologic assessment change of collagen deposition according to H&E staining and the histological grade of fibrosis.PBS group showed thin and wavy collagen, many skin appendages, and a complete subcutaneous fat layer.On the other hand, the untreated lesions (left side of the back) of the BLM group showed thickened and homogenized collagen, thickened dermis, loss of skin appendages, while a thinned subcutaneous fat layer.Of interest, the treated lesions (right side of the back) showed less collagen deposition, thinned dermis, and multiplication of skin appendages in BLM group (Figure 3A).The degree of collagen deposition was considerably higher in the untreated lesions of the BLM group than in the PBS group (p < 0.0001; Figure 3B), demonstrating the successful establishment of a scleroderma mouse model.The degree of collagen deposition on both sides of the back of the PBS group was 1 (p > 0.05; Figure 3C).Compared with the untreated lesions, the treated lesions had a lower degree of collagen deposition in the BLM group (p < 0.001; Figure 3D), indicating that fractional Er:YAG laser treatment ameliorated the fibrosis of scleroderma lesions.

| Fractional Er:YAG laser treatment improved the MMP1 level of mouse morphea model
In order to determine the molecular mechanism, we further analyzed the levels of MMP-1 level and TGF-β1 by immunohistochemical staining.Both the PBS group and the BLM group showed the increased MMP1 level after Er:YAG treatment.Positive staining was found mainly in fibroblast and extracellular matrix.Compared with the untreated lesions (left side of the back), the treated lesions (right side of the back) had more intensive and larger positive area (Figure 4A).The MMP1 expression was considerably reduced in the untreated lesions of the BLM group than in the PBS group (p < 0.05; Figure 4C) and in the BLM group (p < 0.001; Figure 4D), indicating fractional Er:YAG laser treatment upregulated the MMP1 level in morphea lesions.BLM, bleomycin; PBS, phosphate buffer saline.

| Fractional Er:YAG laser treatment improved the TGF-β1 level of mouse morphea model
Both PBS group and BLM group showed limited TGF-β1 level after Er:YAG treatment.Positive staining was found mainly fibroblasts and vascular endothelium.Compared with the untreated lesions (left side of the back), the treated lesions (right side of the back) had less intensive and positive area (Figure 5A).The TGF-β1 level was higher in the untreated lesions of the BLM group than in the PBS group (p < 0.05; Figure 5B).Compared with the untreated lesions, the treated lesions had a lower TGF-β1 level in the PBS group (p < 0.01; Figure 5C) and in the BLM group (p < 0.01; Figure 5D), indicating fractional Er:YAG laser treatment downregulated the TGF-β1 level of morphea lesions.Thus, Er:YAG laser treatment might improve the fibrosis of morphea via modulating TGF-β1/MMP1 axis.

| DISCUSS ION
The results of this study demonstrate that fractional Er:YAG laser is a promising treatment method for morphea mouse model with significant improvements by both objective and subjective measures.
Previous studies have documented marked softening and improved dyschromia after laser treatment. 13,14Shalaby et al. 13 compared the efficacy of CO 2 laser treatment versus low-dose UVA-1 therapy, indicating fractional CO 2 laser improved collagen deposition in morphea patients based on the clinical and histopathologic assessment, which was superior to low-dose UVA-1 phototherapy.
However, the adverse reactions of CO 2 laser treatment, such as erythema, pigmentation and pain are more serious with the higher energy intensity.Compared with CO 2 laser, fractional Er:YAG laser results in effective ablation of target area with minimal tissue ablation and thermal damage to its surrounding tissue, producing less erythema and adverse reaction. 15A recent case showed, marked softening of the sclerotic plaque and improvement in skin coloration and smoothness were noted after fractional Er:YAG laser treatment, without adverse reactions for up to 2 years. 14This study objectively elucidates the improvement in thickness, tissue remodeling, and molecular aspects after fractional Er:YAG laser treatment of morphea mouse model.We demonstrated whether the fractional Er:YAG laser is a beneficial efficacy approach based on clinical, ultrasonic, histopathologic, and immunohistochemical evaluation.The LoSCAT scores, which focuses on clinical manifestation, showed significant improvement in skin thickening and pigmentation.The lower dermis thickness and collagen deposition reflects decreased fibrosis.
The mechanism of fractional laser remodeling on collagen is to remove homogeneous and fibrotic tissue through the formation of MTZs, and a delayed effect of wound healing via collagen production. 19,20primary finding of this study is increased collagen protease MMP1 levels after fractional Er:YAG laser treatment.The upregulation of MMP1 causes the degradation of abnormally homogeneous collagen, thus promoting the replacement of new collagen.Fibrillar collagen is cleaved by select MMPs, including MMP1 and MMP13.21 Previous studies have shown that the expression of MMP1 and MMP3 increased gradually within 3 days after CO 2 laser treatment, reached a peak on day 7, and decreased within 2 weeks.The expressions of MMP9 and MMP13 remain elevated for a longer period of time to ensure the degradation of collagen.22 Another finding in molecular aspects is the significant decrease of profibrotic marker TGF-β1 level after fractional Er:YAG laser treatment.Increased TGF-β1 was observed in the first 3 days after CO 2 laser treatment, peaked on Day 7, and then a gradual decrease over 30 days-6 months, suggesting a physiological controlled wound healing process.23,24 TGF-β1 was upregulated in the early stage after laser treatment to promote wound healing, and downregulated in the late stage to avoid excessive collagen deposition.
Interestingly, we also found there was no statistical differences on clinical, ultrasonic and histopathologic levels in the PBS group, while statistically significant differences were noted in MMP1, and TGF-b1 expression.Such inconsistencies at the molecular and clinical levels might be related to the different states of the collagen been replaced in different groups.The collagen in the PBS group was normal, while the collagen in the BLM group was thickened and homogenized, which is the pathological feature of morphea.
The remodeling process of converting thickened collagen into new This is the first study to prove fractional Er:YAG laser treatment upregulated the MMP1 level, and downregulated the TGF-β1 level in morphea mouse model.The treatment efficacy was proved on clinical, ultrasonic and histopathologic levels, and the results were consistent with those of other studies on the treatment of morphea mouse model with drugs, 25 UVA phototherapy,26,27 and surgery. 28 clear that fractional Er:YAG laser treatment improves morphea greatly.Future studies should address the clinical efficacy of laser treatment; molecular mechanism; dose-response data for the laser treatment; potential synergies of laser with other treatments.
In conclusion, this study proved that fractional Er:YAG laser is an effective treatment for morphea.

AUTH O R CO NTR I B UTI O N S
Hui Xiong and Qing Guo designed the study and edited the manuscript.Junjie Cen performed the experiments and drafted the manuscript.Mingjie He, Danqi Huang, and Zengqi Tang analyzed the data.
All the authors contributed to the overall scientific interpretation and reviewed the manuscript.

2. 10 |
Immunohistochemical evaluation Sections were immune-stained with anti-TGF-β1 (Abcam) and anti-MMP1 (Beijing Biosynthesis Biotechnology Co).Formalin-fixed tissues section was boiled in sodium citrate-EDTA antigen repair solution (Beijing Zhong Shan-Golden Bridge Biological Technology Co) for 10 min and cool down for three times.Antigen visualization was performed using goat anti-rabbit IgG (Beijing Zhong Shan-Golden Bridge Biological Technology Co) and 3,30-diaminobenzidine tetrahydrochlo ride (DAB chromogen) (Beijing Zhong Shan-Golden Bridge Biological Technology Co).Using ×40 objective lens to record immunostaining of TGF-β1 and MMP1 in the random region.Using the Image J analyzer (National Institutes of Health) to quantitative study the TGF-β1 and MMP1 immunohistochemistry.The integratedoption density (IOD) of positive immunoreaction was measured and recorded.

3 | RE SULTS 3 . 1 |
Fractional Er:YAG laser treatment improved the clinical evaluation of mouse morphea model After 2 weeks of applying fractional Er:YAG laser treatment four times, we assessed the thickness, dermal atrophy, dyschromia, and erythema of BLM-induced mouse morphea model according to the adjusted LoSCAT score.The skin on both sides of the back of the PBS group was smooth, soft, and reddish, without nodules or plaques.The untreated lesions (left side of the back) of the BLM group showed typical fibrosis syndromes including round sclerotic plaques, with hair loss and increasing thickness and toughness.The LoSCAT scores were considerably higher in the untreated lesions of the BLM group when compared to those of the PBS groups (p < 0.0001; Figure1A,B), demonstrating the successful establishment of a morphea mouse model.The LoSCAT scores of both sides of the back of the PBS group were 0 (p > 0.05; Figure1A,C).Of note, the lesion (right side of the back) became soft and faded away after Er:YAG laser treatment (Figure1A).The lesions after laser treatment revealed lower LoSCAT scores in the BLM group than untreated lesions (p < 0.01; Figure1D) in the same hosts, indicating that fractional Er:YAG laser treatment reduced the damage caused by morphea lesions.

3. 2 |
Fractional Er:YAG laser treatment improved the ultrasonic manifestations of mouse morphea model Next, we used high-frequency ultrasound to detect the ultrasonic image and dermal thickness associated with the lesions.The ultrasonography of in PBS group showed hyperechoic epidermis and medium-echoic dermis, exhibiting uniform thickness and obvious stratification with a subcutaneous fat layer.In contrast, the ultrasonography of the untreated lesions (left side of the back) of the BLM group showed thickened dermis with a significant impaired subcutaneous fat layer (Figure 2A).In addition, treated lesions (right F I G U R E 1 Fractional Er:YAG laser treatment improved the clinical evaluation of mouse morphea model.(A) Mice were divided into the BLM group (n = 15) and the PBS group (n = 9), subcutaneously injected on their both backside with BLM and PBS, respectively.The right back side were given fractional Er:YAG laser treatment.The left back side had no treatment.Lesions of both backside were imaged after 2 weeks of applying fractional Er:YAG laser treatment.(B) The LoSCAT scores of the untreated lesions (left side of the back) in the BLM group and the PBS group.(C) The LoSCAT scores of the untreated lesions (left side of the back) and the treated lesions (right side of the back) in the PBS group.(D) The LoSCAT scores of the untreated lesions (left side of the back) and the treated lesions (right side of the back) in the BLM group.Each dot represents a mouse.n = 24.ns, no significant difference; **p < 0.01, and ****p < 0.0001.BLM, bleomycin; LoSCAT, Localized morphea Cutaneous Assessment Tool; PBS, phosphate buffer saline.

Figure
Figure 4B).Compared with the untreated lesions, the treated lesions had a increased MMP1 expression both in the PBS group (p < 0.01;

F I G U R E 3
Fractional Er:YAG laser treatment improved the pathologic manifestations of mouse morphea model.(A) The H&E image of both backside lesions section for each group.(B) The degree of collagen deposition of the untreated lesions (left side of the back) in the BLM group and the PBS group.(C) The degree of collagen deposition of the untreated lesions (left side of the back) and the treated lesions (right side of the back) in the PBS group.(D) The degree of collagen deposition of the untreated lesions (left side of the back) and the treated lesions (right side of the back) in the BLM group.Each dot represents a mouse.n = 24.ns, no significant difference; ***p < 0.001, and ****p < 0.0001.BLM, bleomycin; PBS, phosphate buffer saline.

F I G U R E 4
Fractional Er:YAG laser treatment improved the MMP1 level of mouse morphea model.(A) The MMP-1 immunohistochemical staining of both backside lesions section for each group.(B) Quantification of the relative MMP-1 expression of the untreated lesions (left side of the back) in the BLM group and the PBS group.(C) Quantification of the relative MMP-1 expression of the untreated lesions (left side of the back) and the treated lesions (right side of the back) in the PBS group.(D) Quantification of the relative MMP-1 expression of the untreated lesions (left side of the back) and the treated lesions (right side of the back) in the BLM group.Each dot represents a mouse.n = 24.*p < 0.05, **p < 0.01 and ***p < 0.001.BLM, bleomycin; PBS, phosphate buffer saline.collagen brought about decreased in dermal thickness and declined histological grade of fibrosis, which might not be obvious in the remodeling of normal collagen.

3 H/HeN mice Left side back; no treatment (n = 24) Right side back; fractional Er:YAG laser treatment (n = 24)
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