DOI: https://doi.org/10.53350/pjmhs20221611241 ORIGINAL ARTICLE P J M H S Vol. 16, No. 11, November, 2022 241 Formulation of in-Vivo Experimental Model of Oral Submucous Fibrosis in Wistar Rat MUHAMMAD UMAIR PIRACHA 1 , IQRA EJAZ 2 , SAIMA CHAUDHRY 3 , SARAH GHAFOOR 4 1 Department of Oral Pathology, Nishtar Institute of Dentistry, Multan, Pakistan 2 Department of Oral Biology, Bakhtawar Amin Medical and Dental College, Multan, Pakistan 3 Department of Oral Pathology, University of Health Sciences, Lahore, Pakistan 4 Department of Oral Biology, University of Health Sciences, Lahore, Pakistan. Corresponding Author: Iqra Ejaz, Email: iqraejaz094@gmail.com, Cell: 0341 6302433 ABSTRACT Background: Oral submucous fibrosis is a chronic, fibrotic and crippling disease that affects the oral mucosa. It leads to progressive fibrosis that mainly involves the buccal mucosa but other parts of the oral cavity such as the tongue and palate can also be affected. There is a general consensus that areca nut and its constituents are mainly responsible for the pathogenesis of the disease. On histological examination of a biopsy taken from the human oral mucosa, OSMF presents with atrophic epithelium and juxta-epithelial deposition of a vast amount of collagen. Material and Methods:12 healthy male Wistar rats (age 5-6 weeks) were divided into two groups: group A (control) and group B (experimental) after ethical approval. Bleomycin is an anti-cancer drug that is used to create various animal models of fibrosis. In this study, bleomycin was injected into the buccal mucosa of Wistar rat at a concentration of 1mg/mL dissolved in 0.01 M phosphate-buffered saline (PBS) for 4-8 weeks daily. The control group was treated with normal saline. Body weight and mouth openings were recorded. A macroscopic examination was done along with a microscopic analysis of histopathological features and an immunohistochemical analysis of α-SMA expression. Masson’s trichrome stain was used to visualize the collagenous deposition in sub-epithelial tissue. Results: Clinically buccal mucosa showed lesions that mimicked OSMF. Changes in epithelium and lamina propria were observed in the experimental group as compared to the control group. At week 4, stratified squamous epithelium of buccal mucosa showed shortening of rete ridges and lamina propria showed deposition of thick separate collagen. At week 8, epithelium became flat, and atrophic with loss of rete ridges and lamina propria showed juxta-epithelial deposition of collagen which was completely hyalinized and α-SMA expression increased in myofibroblasts. Conclusion: The results from our study suggested that Wistar rats are a reproducible and sustainable model for OSMF. Keywords: Oral submucous fibrosis, animal model, bleomycin, fibrosis, alpha-smooth muscle actin. INTRODUCTION Clinically Oral submucous fibrosis (OSMF) is a chronic disorder of the oral cavity, it presents with limited mouth opening, formation of circumoral fibrotic bands, pale oral mucosa, xerostomia, ageusia, ankyloglossia and ulceration of oral mucosa. (1) OSMF is characterized histo-pathologically into four grades. In grade 1 (very early stage), the overlying epithelium is of normal thickness whereas lamina propria presents with fine fibrillar collagen, strong fibroblastic response and dilated blood vessels. In grade 2 (early stage), epithelium presents with keratinization and shortened rete ridges whereas lamina propria shows thick and separate collagen with dilated vessels and plump fibroblasts. In grade 3 (moderate stage), epithelial atrophy is present with loss of rete ridges and lamina propria exhibits moderate collagen hyalinization, few constricted blood vessels and fibroblasts. In grade 4 (advanced stage), epithelial atrophy occurs with completely hyalinized collagen, obliterated blood vessels and no fibroblastic response in lamina propria. (2) Various triggering components such as genetic disorders, persistent infections, release of inflammatory cytokines and frequent exposure to irritants have been reported in the development of progressive fibrotic disease. (3) High consumption of paan and chaalia has been reported among school children and females with a poor socioeconomic background in India, Pakistan and Asian immigrants based in United states. (4-6) Myofibroblasts are activated fibroblasts which play a crucial role in wound healing, tissue repair and regeneration, maintain tissue homeostasis by regulating the production and degradation of extracellular matrix in tissues after injury. (7-9) Bleomycin was first discovered by Umezewa et al. in 1966, originally isolated from the bacterium Streptomyces verticillus. (10) Bleomycin is an antibiotic medicament, widely used for toxic cellular effects. (11) Previously Zhang et al. created a successful OSMF model in female Sprague-Dawley rats using bleomycin, an anticancer drug, which clinically and pathologically closely resembled grade 4 (advance stage) human OSMF. Clinically the buccal mucosa appeared pale with the formation of fibrous bands. (12) We also tried to replicate the same model that features the characteristics of OSMF and investigate whether the same concentration of bleomycin can induce OSMF in a different strain of rat which is sustainable, reproducible and less time consuming so that we can use it to our advantage to test various treatment modalities that might prove beneficial to humans suffering from OSMF in future. MATERIALS AND METHODS The Ethical Review Committee of University of Health Sciences, Lahore provided guidelines to be followed to conduct study on health male Wistar rats age 5-6 months and weight 150-200 g and approval provided by UHS. Standard rat chow and water ad libitum was given one week prior to the experimental trial. The rats were placed in front of light (6 am -6pm) and in darkness (6 pm- 6 am). This experimental trial divided in to two group as Group A (Control) and Group B (Experimental), comprises of six rats in each assigned group. Normal saline was used in control group and bleomycin was used in experimental group. Both groups were sacrificed at 4 and 8 weeks respectively. A rat model was created using bleomycin (Cipla pharmaceuticals) at a concentration of 1mg/mL dissolved in 0.01 M phosphate buffered saline (PBS). Quantity of 100 μL of bleomycin solution was administered through using a 26-guage brown needle for 4-8 weeks daily into the buccal mucosa of rats. (12) Since week 0 from the beginning of the study body weights and recorded till week 8 of both experimental and control groups. Vernier caliper tool was used to assess the distance of upper and lower incisors teeth. On intra-oral examination of buccal mucosa, changes in color, texture, and flexibility were observed weekly. The animals of both control and experimental groups were immolated at week 4 and week 8 by overdose of chloroform, the day after the final injection of bleomycin. (13) Antigen retrieval was done using 10mM sodium citrate buffer (pH 9) and 0.05% Tween- 20 solution in a water bath at 95°C for 45 min. Positive expression of α-SMA was scored as no expression=(0), 1%-25% of positively staining cells=(1), <25%-50% of positively staining cells=(2) and