Effects of DMSO on a rabbit ear hypertrophic scar model: A controlled randomized experimental study

Accepted Manuscript Effects of DMSO on Rabbit Ear Hypertrophic Scar Model: A Controlled Randomized Experimental Study Elif Sari, MD, Bulent Bakar, Gungor Cagdas Dincel, Fatma Azize Budak Yildiran PII:

S1748-6815(17)30037-2

DOI:

10.1016/j.bjps.2017.01.006

Reference:

PRAS 5216

To appear in:

Journal of Plastic, Reconstructive & Aesthetic Surgery

Received Date: 28 July 2015 Revised Date:

21 October 2016

Accepted Date: 4 January 2017

Please cite this article as: Sari E, Bakar B, Dincel GC, Budak Yildiran FA, Effects of DMSO on Rabbit Ear Hypertrophic Scar Model: A Controlled Randomized Experimental Study, British Journal of Plastic Surgery (2017), doi: 10.1016/j.bjps.2017.01.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Effects of DMSO in Rabbit Ear Hypertrophic Scar Model: A Controlled Randomized Experimental Study

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Running head: DMSO on Hypertrophic Scar

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Elif Sari1, Bulent Bakar2, Gungor Cagdas Dincel3, Fatma Azize Budak Yildiran4

1. Kirikkale University Faculty of Medicine, Department of Plastic, Reconstructive and

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Aesthetic Surgery, Kirikkale, Turkey

2. Kirikkale University Faculty of Medicine, Department of Neurosurgery, Kirikkale, Turkey

3. Aksaray University, Eskil Vocational High School, Laboratory and Veterinary

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Science, Aksaray, Turkey

4. Kirikkale University, Vocational High School Of Health Services, Department of

Correspondence: Elif Sari, MD

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Medical Services and Techniques, Kirikkale, Turkey

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Kirikkale University Faculty of Medicine, Department of Plastic, Reconstructive and Aesthetic Surgery, Ankara- Kirikkale Road 7th km Yahsihan, 71450 Kirikkale- Turkey Telephone: +905063813703 Fax: +90 580 225 28 19 E- Mail: [email protected]

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ACCEPTED MANUSCRIPT Effects of DMSO on Rabbit Ear Hypertrophic Scar Model: A Controlled Randomized Experimental Study Abstract: Dimethylsulfoxide (DMSO) is an anti-inflammatory, anti-bacterial, analgesic drug that is

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widely used to treat several diseases in literature. It has a detractive effect to collagen deposition in abnormal tissue. The aim of this study was to investigate the possible therapeutic effects of the DMSO in the hypertrophic scar formation in rabbit.

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Twenty-four New Zealand male albino rabbits were randomly divided into four groups: control, sham, DMSO, and TRA (triamcinolone acetonide). Except control group, punch

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biopsy defects were created on each animal’s right ear. Following the hypertrophic scar formation at day 28, intralesional DMSO and triamcinolone acetonide was administrated once a week for four weeks into these scars of the DMSO and TRA groups, respectively. No therapeutic agent was administered to the control and sham groups. One week after the last

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injection, ear samples were collected for histopathological, immunohistochemical, and realtime polymerase chain reaction (RT-PCR) gene expression analyses. Histopathological examination revealed that the epithelium in the DMSO group was thicker

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than in the control and TRA groups; but thinner than in the sham group. Connective tissue thickness value and vascularity level value of the sham group was higher than control,

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DMSO, and TRA group values. Collagen type I immunoreactivity level values of the sham and TRA groups was higher than control and DMSO group values. Collagen type III immunoreactivity was higher in sham group than all other groups. Collagen type I/ type III immunoreactivity ratios were lower in DMSO group. Collagen alignments were natural in the DMSO group, but they were irregular in the sham and TRA groups.

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ACCEPTED MANUSCRIPT The collagen type I gene expression level values of the DMSO and TRA groups were lower than the results of the sham group. Collagen type III, and IFN-γ mRNA expression values were almost similar among the groups. TGF-1β mRNA expressions were higher in DMSO and TRA groups than control and sham groups.

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In conclusion, it can be said that intralesional administration of the DMSO could decrease the hypertrophic scar formation easely and safely.

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Key words: Dimethylsulfoxide; DMSO; hypertrophic scar; wound healing; scar

Introduction:

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Today, treatment of the hypertrophic scar during an abnormal wound healing process is a great challenge for physicians. Although various theories (such as ischemia theory1, mast cell theory2, transforming growth factor beta (TGF-β) interaction3, and mechanical theory4) have been discussed in literature, the pathogenesis of hypertrophic scar has not been

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comprehensively determined yet. Before many therapeutic techniques (such as avotermin,5 pressure garments,6 intralesional steroid injection,7 onion extract and heparin gel application,8 silicone gel sheeting,9 bleomycin injection,10 and pulsed dye laser11) have been tested to treat

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the hypertrophic scar occurrence, any of them could decrease or block this process, and no reliable treatment regimen has been identified, yet. Today, intralesional streoid injection just

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has been preferred as a first choice to decrease the hypertrophic scar formation, eventhough it may cause to produce some controversial effects (such as hypopigmentation, subcutaneous fat atrophy, rebound effect, and skin atrophy) in scar tissue.12 Dimethylsulfoxide (DMSO) is a highly lipophilic and hydrophilic anti-inflammatory solvent, and when it is administered to the skin topically, it penetrates the skin immediately.13 It also decreases the pathological deposition of collagen metabolism in fibrotic tissue, but not change to the balance of the normal collagen metabolism in healthy tissue. 14,15 Actually, because of

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ACCEPTED MANUSCRIPT this property, variable concentrations of DMSO gels and creams have been used to treat the scleroderma,16 ischemic ulcers,17 decubitus ulcers,18 and skin necrosis due to antineoplastic agent extravasation19 in human subjects succesfully and this agent was approved as a therapeutic drug by the FDA in 1970.20 But, to the best of our knowledge, the effects of the

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intralesional DMSO to the hypertrophic scar formation have not been investigated in literature, yet. So, present study was constructed to evaluate the possible therapeutic effects of intralesional DMSO in hypertrophic scar tissue in rabbit.

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Materials and Methods: Materials

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This experimental study was performed in accordance with the guidelines for the use of laboratory animal subjects in research set by the Ethical Committee of Ankara Education and Research Hospital (Number: 0019/319).

In this study, 10% DMSO (Micro Therapeutics, Inc., Irvine, CA) diluted with saline solution

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was used intralesionally at a dose of 0.1 ml/day. The density of anhydrous liquid DMSO is approximately 1.1 g/ ml. The cutaneous LD50 for rabbit is 5 g/ kg.21 Triamcinolone acetonide (Kenacort A; Bristol-Myers Squibb, New York, NY) was

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administered intralesionally at a dose of 0.1 ml/day. The density of triamcinolone acetonide is approximately 40 mg/ml. The cutaneous LD50 for rabbit is just over 402 mg/kg.22

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Anesthesia was performed with intraperitoneal administration of 40 mg/ kg ketamine HCl (Ketalar®; Pfizer Inc, USA), and 5 mg/ kg xylazine HCl (Rompun® %2; Bayer HealthCare AG, Germany).

Twenty-four male New Zealand albino rabbits weighting 2250–2500 g were divided randomly into 4 groups 23, as following: •

Control group (Neither surgical procedure nor experimental agent was performed to the animals; n:6), 4

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Sham group (hypertrophic scars were created, but no experimental agent was applied to the animals; n:6),

•

DMSO group (hypertrophic scars were created and 0.1 ml anhydrous DMSO diluted in 0.9 ml saline was injected intralesionally into the scar tissue once a week for 4

•

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weeks; n:6)

TRA group (hypertrophic scars were created and 0.1 ml triamcinolone acetonide was administered intralesionally into the scar tissue once a week for 4 weeks; n:6)

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Surgical Procedure

In present study, the surgical procedure performed to the animals was described by Morris et

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al before and it has been widely used in literature.24 Sedation anesthesia was performed to the animals except the control group. Then the both ears of each rabbit were cleaned by using the betadine solution, and a 5-mm punch biopsy device was used to create a fullthickness punch defect on each ear skin including the perichondrium. After haemosthasis (i.e. manual pressure

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to the punch defect areas), each ear was covered with a sterile bandage. To prevent from microbial contamination, the wound dressings of animals were changed twice a week for 28 day. After the histopathological and macroscopic stigma of the experimental hypertrophic

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scars was observed on day 28, the rabbits were randomly divided into four groups. The

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DMSO group received 0.1 ml diluted DMSO solution administered to scar tissue intralesionally once a week for four weeks, and the TRA group received 0.1 ml of triamcinolone acetonide administered to scar tissue intralesionally once a week for four weeks. The injection was performed using a 28-gauge needle parallel to the center of the scar tissue. One week after the fourth injection, all animals were re-sedated and then scar tissues of the sham, DMSO and TRA groups and normal skin tissue of the control group were collected and each of them was divided into three parts for further laboratory investigation as described following: one-third of the specimen was fixed in 10% formaldehyde for histopathological

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ACCEPTED MANUSCRIPT examination, another one-third was prepared for immunohistochemical analyses, and the remaining part was frozen at –80oC for real-time polymerase chain reaction (RT-PCR) analyses. Finally, all animals were sacrificed by performing the cardiac embolization technique.

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Histopathological Analysis

Scar tissue was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4 for 48 hours and was washed under tap water overnight. Following routine tissue preparation

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procedures, tissue samples were embedded in paraffin blocks. Paraffin serial sections were cut at a thickness of 4–5 µm. Hematoyxlin-eosin (H&E) staining were performed, and the

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sections were analyzed by using a trinocular light microscope (Olympus BX51 and DP25 digital camera). Scar tissue was evaluated according to the scar index obtained from the ratio of hypertrophic scar tissue and normal dermal tissue. When this ratio was greater than 1, it indicated that hypertrophic dermis was obtained. The epithelial thickness, capillary count (i.e.

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vascularity) and connective tissue thickness were counted and analysed in five representative fields of each specimen at 400x magnification. Immunohistochemical Analysis

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The immunostaining techniques were performed to the scar tissue to demonstrate the collagen type I and collagen type III. Commercial antibodies were visualized on 4- to 5-µm-thick

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paraffin sections using an indirect streptavidin/biotin immunoperoxidase kit (HRP; Thermo Scientific, USA). The sections were placed onto adhesive slides, deparaffinized for 5 min. Each in the 3-step xylene series, and rehydrated using a series of graded alcohol and distilled water. The antigens were retrieved by boiling the tissue sections on glass slides in citrate buffer (pH 6.0) (Thermo Scientific, USA) for 20 min. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide in absolute methanol for 7 min at room temperature (RT). The tissue sections were rinsed thrice with PBS (pH 7.4) for 5 min, between each

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ACCEPTED MANUSCRIPT consecutive step. The sections were then incubated in a blocking serum for 5 min to prevent non-specific antibody binding. Thereafter, the sections were incubated with a primary antibody (collagen I and collagen III) for 60 min in a humidity chamber at the RT. After treating the sections with biotin-labeled secondary antibody for 15 min and streptavidin-

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peroxidase enzyme for 15 min at RT, the color reaction was performed using aminoethylcarbasole (AEC) chromogen (Thermo Scientific, USA) for 5–10 min. Sections were counterstained with Mayer’s hematoxylin for 1–2 min and suspended in water-based

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mounting medium (Thermo Scientific, USA). For each immunoperoxidase test, two negative and positive control tissue sections were used. As negative control, the one of the serial

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parafin sections incubated with normal mouse serum (isotype serum control) instead of primary antibody. Additionally, to control non-specific endogenous peroxidase and biotin activities in each test, the primary antibody step was omitted. Quantitative Histomorphometric Analysis

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The density of positive staining was measured using a computerized image system composed of a Leica charge-coupled device camera DFC420 (Leica Microsystems Imaging Solutions, Ltd., Cambridge, UK), connected to a Lecia DM4000 B microscope (Leica Microsystems

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Imaging Solutions, Ltd.). Accordingly, five representative fields were selected under highpower view and consecutive pictures were captured by the Leica QWin Plus v3 software by a

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20× objective (Leica Microsystems Imaging Solutions) at a setting identical to the image system. For examining the staining for each antibody, we used the same setting for all slides. Integrated optical density of all the positive staining of collagen I and collagen III in each photograph was measured. For the quantification of mean was quantified as the collagen Iand collagen III-positive area/total area were measured and calculated on the pictures. All images were collected under the same lighting conditions. To avoid observer bias, all sections were quantified by a blinded investigator. Data were statistically described in terms of mean

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ACCEPTED MANUSCRIPT and standard deviation (mean±SD) for area %. After calculating the proportion (% pixels) of stained area to the whole field, the mean (in % pixels) staining area for each slide was determined.Scar tissue was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4 for 48 hours and was washed under tap water overnight. Following routine tissue

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preparation procedures, tissue samples were embedded in paraffin blocks. Paraffin serial sections were cut at a thickness of 4–5 µm. Hematoyxlin-eosin (H&E) and immunohistochemical staining were performed, and the sections were analyzed.

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Real-Time Polymerase Chain Reaction Analysis

RT-PCR analyses were performed by using gene expression kits (LightCycler 480 SYBR

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Green I Master version 12 in the LightCycler 480, Roche Molecular Biochemicals), according to the manufacturer’s instructions. Collagen type I mRNA, collagen type III mRNA, TGF-1β mRNA, TNF-α mRNA, and IFN-ɣ mRNA expressions were detected. Statistical Analysis

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The values of the epithelial thickness were not normally distributed (Kolmogorov-Smirnov test, p<0.05) and variations were not homogenous among the groups and within the groups

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(Levene’s test, p<0.05). Therefore, all results were statistically analyzed by using the Krusskal-Wallis Multiple Variant Analysis test, and p values less than 0.05 were considered to

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be significant. The determination of the statistical differences (post hoc evaluation) between the groups, the Mann-Whitney U test, and the Bonferroni correction test were performed to all results, and p values lower than 0.0083 were considered to be significant. The capillary count (vascularity) values, connective tissue thickness (matrix) values, collagen type I, and collagen type III immunoreactivity level values, collagen type I, collagen type III, TGF-1β, IFN-γ and TGF-1β gen expression values were normally distributed (KolmogorovSmirnov test, p>0.05), and variations of them were homogenous among the groups and within the groups (Levene’s test, p>0.05). Therefore, the One-Way Analysis of Variance (ANOVA) 8

ACCEPTED MANUSCRIPT test was performed for all these values; and the p values of less than 0.05 were considered to be significant. To determine the statistical differences between the groups, post hoc evaluation (One-Way ANOVA-Tukey HSD Multiple Comparisons test and the Bonferroni correction test) test was performed. The p values of less than 0.0083 were considered to be significant.

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Results: Macroscopic Findings

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Neither local inflammation nor abscess was observed at the surgical area of the rabbits. All wounds were completely epithelized by the second week and hypertrophic scars were formed

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in all groups by the day 28.

During the macroscopic inspection of the scars, there was a noticeable difference in scar quality between the groups. The scars were much more smooth and rough in the DMSO group than the TRA and sham groups (Figure 1).

Epithelial thickness:

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Histopathological Findings

Epithelial thickness values were statistically different between the control and sham (Z=-

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2.882, p=0.004), control and DMSO (Z=-2.882, p=0.004), control and TRA (Z=-2.647, p=0.008), sham and DMSO (Z=-2.882, p=0.004), sham and TRA (Z=-2.882, p=0.004), and

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DMSO and TRA (Z=-2.882, p=0.004) groups. The epithelium in the DMSO group was thicker than in the TRA and control groups, but more thinner than in the sham group. (Table 1, Table 2, Table 3).

Connective tissue thickness: Connective tissue thickness values were statistically different between the control and sham (p<0.001), control and DMSO (p=0.006), sham and DMSO (p<0.001), and sham and TRA (p<0.001) groups. Connective tissue thickness value of the sham group was higher than control, DMSO, and TRA group values (Table 1, Table 4, and Table 5). In spite of the TRA 9

ACCEPTED MANUSCRIPT and sham groups, the collagen alignments were observed natural and mature in the DMSO group, as well as control group. Vascularity: The vascularity level values were different between the control and sham (p<0.001), and

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control and TRA groups (p<0.001) but not different between the control and DMSO (p=0.123) groups, statistically. Additionally, the mean vascularity level value of the TRA group was almost smilar to the sham group value (p=0.011), and vascularity level values of

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both groups were higher than control and DMSO group values (Table 1, Tables 4, and, Table

Immunohistochemical findings Immunoreactivity of the collagen type I:

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5).

The collagen type I immunoreactivity level values were statistically different between the control and sham (p<0.001), sham and DMSO (p<0.001), sham and TRA (p<0.001), and

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DMSO and TRA (p=0.002) groups; but not different between the control and DMSO (p=0.856), and control and TRA (p=0.009) groups. Collagen type I immunoreactivity level

Table 5).

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was higher in sham and TRA groups than in control and DMSO groups (Table 1, Table 4 and

Immunoreactivity of the collagen type III:

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The collagen type III immunoreactivity level values were statistically different between control and sham (p<0.001), sham and DMSO (p<0.001), sham and TRA (p<0.001) groups; but not different between the the control and DMSO (p=0.491), control and TRA (p=0.221), and DMSO and TRA (p=0.942) groups. Collagen type III immunoreactivity was higher in sham group than all other groups. Furthermore, collagen type III immunoreactivity level value was lower in DMSO group than in TRA group numerically, although there was no significant difference between DMSO and TRA groups (Table 1, Table 4 and Table 5).

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ACCEPTED MANUSCRIPT RT-PCR findings Collagen type I: The collagen type I gene expression values were statistically different between the control and sham (p<0.001), sham and DMSO (p=0.006), and sham and TRA (p<0.001) groups; but not

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different between the control and DMSO (p=0.028), control and TRA (p=0.993), and DMSO and TRA (p=0.184) groups (Table 6, Table 7, and Table 8). Collagen type I mRNA expression level values of the DMSO and TRA groups were lower than the sham group value

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but not different from the control group value. Collagen type III:

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Relative values of collagen type III mRNA level were not different among the groups (Table 6, Table 7, and Table 8). IFN-γ:

The IFN-γ gene expression values were almost equal in all groups (Table 6, Table 7).

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TGF-1β:

The TGF-1β gene expression values were statistically different between the control and DMSO (p<0.001), control and TRA (p<0.001), sham and DMSO (p<0.001) and sham and

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TRA (p<0.001) groups; but not different between the control and sham (p=0.883), and DMSO and TRA (p=0.976) groups (Table 6, Table 7, and Table 8).

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TNF-α:

In the test groups, any amplification was observed for TNF-α relative values. Discussion:

Histopathological results The main aim of present study was to investigate the possible therapeutic effects of intralesional administration of the DMSO and triamcinolone acetonide in hypertrophic scar tissue formation in rabbit model. Actually, study results demonstrated that sham group had

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ACCEPTED MANUSCRIPT higher epithelial and matrix thicknesses, type I and III collagen depositions, and vascularity level values than the DMSO and TRA groups. On the other hand, despite the steroidal agent, DMSO could well decrease the hypertrophic scar tissue without increasing skin and connective tissue atrophy, although epithelization was completed in all groups. Furthermore,

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the collagen alignments in the DMSO group were as natural and mature as in control group. However, they were irregular in the TRA and sham groups. Moreover, the vascularity level value of the DMSO group was lower than sham and TRA group values. This evidence may be

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caused from the finishing of the neovascularization process at the end of the fourth week of the injury before DMSO administration. This paler scar tissue in DMSO group could be

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associated with the lower vascularity levels. On the other hand, the capillary count values of the TRA and sham groups were almost equal, and it could be said that triamcinolone acetonide could not reduce the red color in the scar tissue in rabbit, efficiently. Both collagen type I and type III increases during the wound-healing process and decrease

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during the remodeling phase.25 It has been known that ratio of the collagen type I to collagen type III increases during the remodeling phase, however this ratio decreases in hypertrophic scars.26-28. In present study, collagen type I immunoreactivity level was higher in sham and

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TRA groups than in control and DMSO groups. Furthermore, collagen type I mRNA expression level values of the DMSO and TRA groups were lower than the sham group value;

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but not different from the control group value. Moreover, collagen type III immunoreactivity level value was higher in sham group than in all other groups values; and collagen type III immunoreactivity level value of the DMSO group was lower than TRA group value, numerically, although there was no significant difference between DMSO and TRA groups, statistically. These results could be revealed that DMSO could be effective on scar maturation. Real time PCR results

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ACCEPTED MANUSCRIPT In this study, the collagen type I mRNA expression level value increased in the sham group. However, the mRNA expression level values were similar in the DMSO and TRA groups. These results showed that DMSO could have a beneficial therapeutic effect in the treatment of hypertrophic scar in rabbit. The collagen type III mRNA expression level value was lower in

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the DMSO group than in the sham group. This result could be associated with the idea that DMSO could effectively decrease the collagen type III mRNA expression in hypertrophic scar tissue in rabbit. This type of collagen is immature, which is a characteristic of

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hypertrophic scar tissue.

Ferguson5 reported that TGF-1β is a fibrogenic cytokine and it links to the plasminogen

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activator inhibitor 1 pathway which stimulates matrix synthesis. In addition, Rockwell et al. 4 drew attention to the mechanical tension occurring at the cellular level which causes fibrogenesis. TGF-1β-mediated signaling pathways are very complex and believed to be closely associated with hypertrophic scarring.26 Smad2 and Smad3 predominantly mediate

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signals from activated TGF-1β receptors.27 Although these two receptors are homologous, each of them play different roles in TGF-1β signaling.28 While Smad2 accelerates extracellular matrix production, Smad3 decreases it.29-35 Although TGF-1β gene expression

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level values of the sham, DMSO, and TRA groups were measured higher than control group value in present study, it was observed that DMSO could significantly decrease the collagen

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synthesis in hypertrophic scars of rabbits. So, to explain the effect of DMSO to the TGF-β pathways should be closely investigated in further studies. IFN-γ is known as a repressive cytokine in hypertrophic scar formation, and it has a negative effect in the TGF-1β signaling pathway and collagen synthesis.

29-31

Present study results

demonstrated that there was no significant difference among the groups in terms of IFN-γ mRNA expression, statistically. Therefore, it can be hypothesized according to these results

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ACCEPTED MANUSCRIPT that DMSO could decrease the hypertrophic scar formation in rabbit by the downregulation of TGF-β signaling rather than IFN-γ over-expression. This study had several limitations. First, this study was conducted on the rabbit ear model and the injections were administered once a week for four weeks to the animals. Second, the

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study results could not be extrapolated to the clinical scenario. So, further clinical studies should be designed in the future. Conclusion:

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In conclusion, to reduce the hypertrophic scar tissue formation without skin and subcutaneous

method than streoid applications. Conflict of interest: None Acknowledgment:

Financial Disclosure:

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None

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tissue atrophy, intralesional DMSO administration could be simple, quick, safe, and effective

References:

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The study was funded by the “Scientific Research Project Fund” of Kirikkale University.

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ACCEPTED MANUSCRIPT 32. X Guo, XF Wang. Signaling cross-talk between TGF-beta/ BMP and otherpathways. Cell Res. 2009; 19: 71-88. 33. CH Heldin, K Miyazono, P ten Dijke. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 1997; 390: 465-471.

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34. K Yanagisawa, H Osada, A Masuda et al. Induction of apoptosis by Smad3 and downregulation of Smad3 expression in response to TGF-beta in human normal lung epithelial cells. Oncogene 1998; 17: 1743-1747.

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35. Wang X, Chu J, Wen CJ et al. Functional characterization of TRAP1-like protein involved in modulating fibrotic processes mediated by TGF-β/Smad signaling in

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hypertrophic scar fibroblasts. Exp Cell Res. 2015; 332: 202-211. Figure Legends:

Figure 1: Macroscopic features of the scar tissues (a) in sham, (b) in DMSO, and (c) in TRA groups, respectively.

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Figure 2: (a) Mild expression of collagen type I in normal skin. ABC technique (anticollagen type I), Mayer's hematoxylin counterstain, Bar, 100 µm. (b) Mild expression of collagen type III in normal skin. ABC technique (anti-collagen type III), Mayer's hematoxylin

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counterstain, Bar, 100 µm. (c) Mild expression of collagen type I in DMSO group. ABC technique (anti-collagen type I), Mayer's hematoxylin counterstain, Bar, 100 µm. (d) Mild

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expression of collagen type III in DMSO group. ABC technique (anti-collagen type III), Mayer's hematoxylin counterstain, Bar, 100 µm. (e) Moderate/severe expression of collagen type I in the TRA group. ABC technique (anti-collagen type I), Mayer's hematoxylin counterstain, Bar, 100 µm. (f) Strong expression of collagen type III in TRA group. ABC technique (anti-collagen type III), Mayer's hematoxylin counterstain, Bar, 100 µm. (g) Moderate/severe expression of collagen type I in sham group. ABC technique (anti-collagen type I), Mayer's hematoxylin counterstain, Bar, 100 µm. (h) Strong expression of collagen

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ACCEPTED MANUSCRIPT type III in sham group. ABC technique (anti-collagen type III), Mayer's hematoxylin counterstain, Bar, 100 µm. Table Legends: Table 1: Descriptive table of the histopathological and immunohistochemical results (SD:

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Standart Deviation)

Table 2: Comparison of epithelial thicknesses of the groups (Krusskal-Wallis Multiple Variant Analysis test. p<0.05)

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Table 3: Comparison of the epithelial thicknesses values (Mann Whitney-U test, and

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Bonferroni correction test. p<0.0083)

Table 4: Comparison of vascularity, connective tissue thickness, type I collagen immunoreactivity, and type III collagen immunoreactivity level values of the groups (OneWay Analysis of Variance (ANOVA) test. p < 0.05)

Table 5: Comparison of vascularity, connective tissue thickness, type I collagen

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immunoreactivity, and type III collagen immunoreactivity level values (One- Way Analysis of Variance (ANOVA) test, Tukey HSD Multiple Comparisons test, and Bonferroni correction

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test. p<0.0083)

Table 6: Descriptive table of the real time-PCR gene expression level results (SD: Standart

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Deviation)

Table 7: Comparison of Type I collagen, Type III collagen, IFN-γ and TGF-1β gene expression results of the groups (One-Way Analysis of Variance (ANOVA) test. p<0.05) Table 8:

Comparison of Type I collagen, Type III collagen, IFN-γ and TGF-1β gene

expression level results (One- Way Analysis of Variance (ANOVA) test, Tukey HSD Multiple Comparisons test, and Bonferroni correction test. p < 0.0083)

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ACCEPTED MANUSCRIPT Table 1 Mean/ Median*

SD

29.49

21.15

3.95

195.41

221.05

208.18

10.93

Vascularity

0.00

2.00

Collagen type I

1.75

3.21

Collagen type III

2.25

3.75

Epithelial thickness*

343.86

364.91

Connective tissue thickness

987.21

Vascularity

10.00

Epithelial thickness*

18.24

Connective tissue thickness

2.34

0.49

2.70

0.54

356.62

7.56

1216.46

1092.47

88.62

15.00

12.67

1.75

4.81

4.32

0.42

3.69

4.40

4.10

0.24

57.48

61.31

59.29

1.59

289.26

363.93

312.93

27.89

1.00

4.00

2.50

1.05

Collagen type I

1.95

2.40

2.17

0.17

Collagen type III

2.70

3.41

2.97

0.24

Epithelial thickness*

27.65

31.02

30.18

1.28

259.48

327.72

295.46

23.54

9.00

12.00

10.17

1.17

Collagen type I

2.69

3.32

3.07

0.24

Collagen type III

2.90

3.26

3.08

0.14

Epithelial thickness*

Connective tissue thickness

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Vascularity

Connective tissue thickness Vascularity

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TRA

0.75

3.77

Collagen type III DMSO

0.83

EP

Collagen type I

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Control

Sham

Minimum Maximum

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Variables

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df

P

21.264

3

<0.001

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Epithelial thickness

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Control/ Sham

-2.882

0.004

Control/ DMSO

-2.882

0.004

Control/ TRA

-2.647

0.008

Sham/ DMSO

-2.882

0.004

Sham/ TRA

-2.882

0.004

DMSO/ TRA

-2.882

0.004

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Table 4

df

F

Connective tissue thickness

3

439.273

<0.001

Vascularity

3

130.592

<0.001

Collagen type I

3

45.057

<0.001

Collagen type III

3

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p

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GROUPS (I/J)

Connective tissue thickness Mean p Difference (I/J)

Vascularity

Collagen type I

Collagen type III

Mean Difference (I/J)

p

Mean Difference (I/J)

p

Mean Difference (I/J

p

-884.291

<0.001

-11.833

<0.001

-1.980

<0.001

-1.402

<0.001

Control/ DMSO

-104.747

0.006

-1.667

0.123

0.163

0.856

-0.273

0.491

Control/ TRA

-87.278

0.025

-9.333

<0.001

-0.735

0.009

-0.380

0.221

Sham/ DMSO

779.545

<0.001

10.167

<0.001

2.143

<0.001

1.128

<0.001

Sham/ TRA

797.013

<0.001

2.500

0.011

DMSO/ TRA

17.468

0.922

-7.667

<0.001

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Control/Sham

<0.001

1.022

<0.001

-0.898

0.002

-0.107

0.942

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EP AC C

TRA

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Maximum 9.83 0.88 0.22 76.21 85.06 5.26 0.25 26.14 36.39 0.74 0.31 250.30 3.96 0.14 0.50 344.90

Mean 4.70 0.41 0.16 45.51 49.95 2.18 0.13 21.77 19.22 0.43 0.17 203.36 2.63 0.06 0.34 216.63

SD 3.21 0.30 0.09 18.99 22.17 1.88 0.10 4.06 10.05 0.22 0.12 34.68 1.14 0.60 0.20 94.64

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DMSO

Minimum 1.00 0.10 0.00 24.82 26.00 0.48 0.00 15.22 11.91 0.14 0.00 167.80 1.02 0.00 0.00 105.80

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Sham

Variable Collagen type I Collagen type III IFN-γ TGF-1β Collagen type I Collagen type III IFN-γ TGF-1β Collagen type I Collagen type III IFN-γ TGF-1β Collagen type I Collagen type III IFN-γ TGF-1β

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Table 7

p

Collagen type I

15.784

<0.001

Collagen type III

4.960

0.013

IFN-γ

2.497

0.097

TGF-1β

19.913

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F

<0.001

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ACCEPTED MANUSCRIPT Table 8 Collagen type I

0.045

Mean Difference (I/J) 23.740

0.883

-14.520

0.280

-0.020

1.000

-157.865

<0.001

Control/ TRA

2.075

0.993

0.357

0.934

-171.115

<0.001

Sham/ DMSO

30.730

0.006

1.750

0.048

-181.605

<0.001

Sham/ TRA

47.325

<0.001

2.127

0.014

-194.855

<0.001

DMSO/ TRA

16.595

0.184

0.377

0.924

-13.250

0.976

p

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Control/ DMSO

p

p

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<0.001

Mean Difference (I/J) -1.769

Control/Sham

Mean Difference (I/J) -45.250

TGF-1β

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GROUPS (I/J)

Collagen type III

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