|Year : 2015 | Volume
| Issue : 1 | Page : 11-18
Sprague-Dawley rats are a sustainable and reproducible animal model for induction and study of oral submucous fibrosis
Shilpa Maria, VV Kamath, PS Krishnanand, R Komali
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Bangalore, India
|Date of Web Publication||20-May-2015|
Prof. V V Kamath
Dr. Syamala Reddy Dental College Munnekolala, Marathalli, Bangalore
Source of Support: None, Conflict of Interest: None
Background: Oral submucous fibrosis (OSF) is a chronic debilitating disease predominantly affecting the oral cavity and oropharynx. Characteristic histological traits of OSF include epithelial atrophy, inflammation, and a generalized submucosal fibrosis. Several studies and epidemiological surveys provide substantial evidence that areca nut is the main etiological factor for OSF. Hesitance of patients to undergo biopsy procedure together with clinicians becoming increasingly reluctant to take biopsies in cases of OSF has prompted researchers to develop animal models to study the disease process. Materials and Methods: The present study evaluates the efficacy, sustainability, and reproducibility of using Sprague-Dawley (SD) rats as a possible model in the induction and progression of OSF. Buccal mucosa of SD rats was injected with areca nut and pan masala solutions on alternate days over a period of 48 weeks. The control group was treated with saline. The influence of areca nut and pan masala on the oral epithelium and connective tissue was evaluated by light microscopy. Results: Oral submucous fibrosis-like lesions were seen in both the areca nut and pan masala treated groups. The histological changes observed included: Atrophic epithelium, partial or complete loss of rete ridges, juxta-epithelial hyalinization, inflammation and accumulation of dense bundles of collagen fibers subepithelially. Conclusions: Histopathological changes in SD rats following treatment with areca nut and pan masala solutions bears a close semblance to that seen in humans with OSF. The SD rats seem to be a cheap and efficient, sustainable and reproducible model for the induction and development of OSF.
Keywords: Areca nut, experimental model, oral submucous fibrosis, pan masala, Sprague-Dawley rats
|How to cite this article:|
Maria S, Kamath V V, Krishnanand P S, Komali R. Sprague-Dawley rats are a sustainable and reproducible animal model for induction and study of oral submucous fibrosis. J Orofac Sci 2015;7:11-8
|How to cite this URL:|
Maria S, Kamath V V, Krishnanand P S, Komali R. Sprague-Dawley rats are a sustainable and reproducible animal model for induction and study of oral submucous fibrosis. J Orofac Sci [serial online] 2015 [cited 2019 Dec 9];7:11-8. Available from: http://www.jofs.in/text.asp?2015/7/1/11/157364
| Introduction|| |
Oral submucous fibrosis (OSF) first reported in 1952  is a chronic insidious disease affecting the oral cavity and sometimes the pharynx. It is associated with juxta-epithelial inflammatory reaction followed by a fibroelastic change of the lamina propria, with epithelial atrophy leading to the stiffness of the oral mucosa, trismus, reduced mouth opening and inability to eat.  The development and progression of OSF are closely associated with adverse habits like chewing areca nut and its commercially popular forms such as pan masala and gutka. 
Areca nut which is the basic ingredient of a variety of widely used chewing products is the fruit of the oriental palm Areca catechu. The seed which is enclosed within a fibrous husk is slightly bitter and has a characteristic astringent taste. Depending on individual preference, the nut may be consumed whole, as thin slices or after processing.  The commercial forms of areca nut called "pan masala (without tobacco)" and "gutka (with tobacco)" have flourished in the Indian markets following aggressive marketing by the manufacturers. These products are said to contain high concentrates of areca nut per chew and appear to cause OSF. They are inexpensive and affordable owing to lower taxes imposed on them, thus easily accessible even to minors.
The study of fibrosis is the mainstay in understanding the pathogenesis of the lesion. The debility caused by the lesion includes trismus and inability to consume spicy food. The potential for malignant transformation is one of the primary reasons for monitoring the condition.  Understanding tissue processes and detecting development of malignancy necessitates a biopsy, a procedure feared by patients and surprisingly hesitated by clinicians. Using animal models, thus becomes a crucial and sometimes the only way to study the disorder because such intricacy cannot be duplicated in cell cultures or nonliving systems. Ethical issues notwithstanding, scientists all over the world have acknowledged the importance of the use of animals in several areas of research. They have been used effectively to decipher the etiology, pathogenesis, and treatment outcome of several diseases. 
The Sprague-Dawley (SD) rat was first produced at SD farms, now a corporation called Harlan SD, and is widely used due in part to its calmness and ability to be handled easily. However, characteristic of the species, the rat will still use its sharp incisors to bite if it is handled improperly. SD rats can live up to 3.5 years and grow to an adult body weight of 250-300 g for females and 450-520 g for males. The breed also possesses a number of remarkable anatomical features. First, these rats are unable to vomit due to the placement of the esophagus as it enters the stomach. Furthermore, these rats have no gall bladder, as well as a peculiar lung arrangement: The left lung has one lobe while the right lung has four. Most interestingly, SD rats produce secretions from their eyes when stressed that contain a pigment which, when dry, has the appearance of dried blood. These "tears" glow fluorescently under ultraviolet light. Both females and males become sexually mature at about 65 days old, and the rats can breed throughout the entire year. The gestation period of the SD is only 22 days, and litters can consist of up to 12 pups. Pups can be weaned at around 3 weeks, and females can begin cycling again only 2-4 days after weaning. ,
In the present study, we have successfully developed an animal model using SD rats for induction of OSF following application of areca nut and pan masala extracts. A histological comparison with human OSF cases of varying grades was made, and image analysis was used to create a grading system for the first time in experimental animals.
| Materials and Methods|| |
Healthy SD rats [Figure 1] weighing 120-150 g were chosen for the study. Thirty rats were divided into three groups: Areca nut group-10, pan masala group-10, and control-10. They were kept in clean, hygienic cages and maintained under standard laboratory conditions. The rats were kept in groups of five per cage at controlled temperature (25°C ± 2°C) with 12 h light/dark cycle and humidity. They received standard diet and water ad libitum.
Ethical permission for undertaking this study was obtained from Institutional Animal Ethics Committee. The animals were maintained in accordance with Committee for the Purpose of Control and Supervision of Experimental Animals guidelines for the care and use of laboratory animals.
The areca nut extract was prepared by dissolving 0.2 g of powdered areca nut in 6 ml of distilled water. It was then centrifuged at 15000 rpm for 30 min. The supernatant was collected and used for injection into the rat buccal mucosa. Similar protocol was followed for the preparation of pan masala solution. Though many commercial brands are available in the market, the contents of most are unknown due to nondisclosure by the manufacturers on the wrappings. The present study used a commercial brand called "super" which had listed contents as (betel nuts, catechu, permitted spices, saffron, cardamom, lime, menthol, and added flavors).
Experimental rats in the areca nut and pan masala groups were injected with 0.2 ml of the prepared areca nut and pan masala extracts, respectively, on every alternate day for 48 weeks. Rats in the control group were injected with 0.2 ml of sterile saline. The site of injection was the right buccal mucosa.
Animals from each group were randomly sacrificed at an interval of every 6 weeks. The rats were killed with an overdose of chloroform. The right buccal mucosae were dissected out. The tissues were fixed in 10% formalin followed by conventional processing, sectioning, and Hematoxylin and eosin (H and E) staining for histopathological assessment. The H and E stained sections were also subjected to image analysis by using BIOLUX ® software (Lawrence and Mayo Inc., India). The amount of fibrosis was measured in microns.
The H and E stained sections were initially graded according to Pindborg and Sirsat classification,  into Grade I, II, and III. The values obtained from the image analysis were used to categorize the samples into grades similar to that used for humans. Thereafter, we compared the two grading systems.
| Results|| |
Monitoring clinical changes by visual examination were hindered owing to the small size of the rat oral cavity. Assessment of the H and E stained tissue sections under the light microscope showed OSF-like lesions in different degrees in both the areca nut- and pan masala-treated groups. The histologic changes observed included atrophic epithelium, partial or complete loss of rete ridges, presence of inflammatory cells, juxta-epithelial hyalinization, and accumulation of dense bundles of collagen fibers in the lamina propria. Although the changes were not uniformly progressive, our findings bear a close semblance to the histological traits of OSMF as seen in humans and documented in the literature.  [Table 1] gives a detailed description of histopathological changes observed at every 6 weeks, beginning from the 6 th week and extending up to the 48 th week. [Table 2] and Graph 1 provide the extent of fibrosis measured in microns, as obtained from the image analysis software [Figure 2], [Figure 3], [Figure 4].
|Figure 2: Composite of three photomicrographs showing effects on the buccal mucosa of SD rats at 6 weeks injected with (a) control (normal saline) (b) areca nut (c) pan masala. Note the hyperplasia of the epithelium in (a) and (b) while (c) shows atrophy and initial fibrosis (H and E, original magnification ×10)|
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|Figure 3: Composite of three photomicrographs showing effects on the buccal mucosa of SD rats at 12 weeks injected with (a) Control (normal saline) (b) areca nut (c) pan masala. Note the persistent epithelial with no fibrosis in (a), while (b) and (c) show fibrosis of the submucosa and epithelial atrophy (H and E, original magnification ×10)|
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|Figure 4: Composite of three photomicrographs showing effects on the buccal mucosa of SD rats at 12 weeks injected with (a) Control (normal saline) (b) areca nut (c) pan masala. Note that the control shows regression of the hyperplasia of epithelium and normal submucosa while both (b) and (c) show advanced fibrosis and epithelial atrophy (H and E, original magnification ×10)|
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|Table 1: Histopathological changes observed in the experimental groups of SD rats|
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|Table 2: Quantification of fibrosis from the image analysis data and segregation into grades based on statistical calculations|
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In the areca nut extract group, the amount of fibrosis reached its peak following 18 weeks of treatment. The least amount of fibrosis was seen at the end of 6 weeks. Whereas in the pan masala group, the maximum amount of fibrosis was seen following 24 weeks of treatment. The least amount of fibrosis was seen at the end of 6 weeks.
In the absence of any established grading system for the rat model of OSF, one was created in this particular study. This helped us correlate and compare the changes with human data.
The H and E stained sections were initially graded according to Pindborg and Sirsat classification  into Grade I, II, and III. Among the total of 24 samples, 8 samples showed Grade I fibrosis, 11 samples showed Grade II fibrosis, and 5 samples presented with Grade III fibrosis.
The values obtained from the image analysis [Table 2] were used to categorize the samples into grades similar to that used for humans. Accordingly, fibrosis derived through a linear measurement was standardized statistically into 3 groups: Grade I (107.325 ± 28.01 μ), Grade II (190.328 ± 49.28 μ) and Grade III (271.59 ± 62.81 μ).
[Table 3] compares the grading obtained from Pindborg and Sirsat grading system of OSMF with the grading derived from the image analysis data. Grades obtained from the image analysis data are similar to grades obtained from the Pindborg Sirsat grading system of OSMF in almost all the samples. A variation was observed in the following samples: 30 th week control (167.86 μ), 48 th week control (250.62 μ), 6 th week areca nut (132.16 μ), 42 nd week areca nut (263.09 μ), 48 th week areca nut (286.74 μ), 30 th week pan masala (202. 38 μ), 48 th week pan masala (244.28 μ). But the variation in grading occurred only by a marginal difference from the range that we arrived at from the statistical analysis.
|Table 3: Comparative analysis of Pindborg's grading system (humans) versus grading based on image analysis (rats) as applied to the present study|
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| Discussion|| |
Classical histological features of OSF include atrophic epithelium, juxta-epithelial hyalinization, deposition of collagen in different densities and inflammatory cell infiltration. Despite the fact that changes in the epithelium are variable, most cases show generalized and conspicuous atrophy of the oral epithelium with the loss of rete ridges.  The results obtained from our study showed OSF-like lesions in different degrees in both the areca nut- and pan masala-treated groups. The histologic changes observed included atrophic epithelium, partial or complete loss of rete ridges, inflammation, accumulation of dense bundles of collagen fibers, juxta-epithelial hyalinization. Our findings bear a close resemblance to the characteristic features of OSMF as seen in humans and also as recorded in the literature. 
In the present study, monitoring clinical changes by visual examination was hindered owing to the small size of the rat oral cavity. However, on the histological evaluation, a significant amount of fibrosis was noted in both the areca nut and pan masala groups, a finding which is again consistent with the pathognomic histopathological feature of OSF. The fibrosis that developed in the rats parallels those seen in human samples. Mucosal atrophy is an important feature of OSF subsequent to areca nut chewing for several years.  Our study showed a characteristic atrophic epithelium in the buccal mucosa of all the experimental rats in the areca nut and pan masala groups at every stage of assessment (interval of every 6 weeks) with the pan masala group showing atrophy as early as in the 6 th week.
In our study, areca nut group showed fibrosis beginning in the 6 th week and in the pan masala group fibrosis was evident from the 12 th week forward. A maximum peak in fibrosis was noted in the mid weeks (18 th -30 th week). The histological changes manifested in the SD rats at different time intervals following treatment with areca nut and panmasala solutions were not uniformly progressive. Differences in species, size of tissue, and response to inciting stimuli may have caused a variation in the expression in the tissues.
The fibrosis which developed in the test groups subsequent to the application of areca nut and pan masala solutions may be an attempt to repair the damage caused by irritation of tissue to these test solutions. This kind of pathological situation is most likely to occur with a mild irritation for long periods resulting in a chronic inﬂammatory manifestation in the tissues affected.  In an earlier study,  the application of capsaicin on the palates of Wistar rats for long periods earlier failed to bring about clinical or histological changes consistent with that of OSF. It is to be noted the induction of fibrosis upon application of agents is different from the condition of OSF. The effect of individual components of the betel quid, when applied in isolation, may or may not induce histological features of the condition. A true replication of the human model would be an application in toto of the mixture as it is consumed. A special mention needs to be made regarding arecoline. This alkaloid of the betel nut has been conclusively proven to be fibrogenetic and forms the main ingredient that causes the condition. In the present study, the mild irritation caused by injection of areca nut and pan masala solutions into rat buccal mucosa produced tissue-speciﬁc responses which may be attributed to the components of areca nut and pan masala solutions.
The ingredients used for induction of OSF in animal models have ranged from capsaicin, , lime,  arecoline  to aqueous extracts of areca nut, ,, pan masala, , and gutka (areca nut + tobacco). , Pan masala is a relatively new product in the Indian market, and literature shows only two studies , having attempted application of the same with comparable results. The type of animal model used differs from region to region and is probably based on availability and previous experiences of the investigator. The most common have been albino mice, Wistar rats, and SD rats. [Table 4] lists the studies that attempted to induce OSF in experimental animals using various materials.
Interestingly, the pan masala treated group showed atrophic changes in the epithelium beginning from the 6 th week itself, with the lamina propria showing no evidence of fibrosis. Subepithelial fibrosis was noticeable only from the 12 th week onward. On the other hand in the areca nut treated group, thinning of epithelium was evident only after 12 weeks, with atrophic changes in the epithelium manifested predominantly following 18 weeks of treatment. However, fibrosis in the subepithelial region was noticeable from the 6 th week. Areca nut extracts and arecoline have been shown to be cytotoxic to epithelial cells. The cell death induced by arecoline has been shown to be due to reactive oxygen species production due to suppression of catalase activity in the epithelium.  Thus, areca nut-induced cytotoxicity on epithelial cells could be mediating epithelial atrophy, a hallmark of OSMF.
The early manifestation of atrophic epithelium in the pan masala-treated group could be due to the combined effects of areca nut and other deleterious agents in pan masala such as polycyclic aromatic hydrocarbons, nitrosamines, toxic metals, and pesticide residues. ,
Canniff and Harvey in 1981  observed that treatment of human fibroblasts with areca nut crude extracts increases collagen production. Furthermore, evidence has been provided to support that areca nut extracts decrease collagen phagocytosis by fibroblasts and increase the collagen stability rendering them resistant to collagenase activity. , Areca nut is an essential ingredient of pan masala. Hence, the induction of fibrosis in both the areca nut- and pan masala-treated groups could be due to the proliferative action of areca nut on the fibroblasts.
As evident from the results of our study, the extent of fibrosis in the animals injected with saline was minimal when compared to the amount of fibrosis seen in the areca nut- and pan masala-treated groups, except in the terminal weeks. The control group which was injected with saline showed noticeable fibrosis in the 42 nd and 48 th week. The fibrosis that developed was probably a reparative response to tissue irritation following the act of injection over a prolonged period of time. However, epithelial atrophy, the hallmark of OSF was not manifested by any of the controls. Thus, implying that the changes manifested in the areca nut and pan masala groups was definitely due to the deleterious effect of the constituents in the areca nut and pan masala solutions.
When attempts to extrapolate the data from our study to the development of OSMF in humans were made interesting observations were recorded. The average life span of humans is about 70 years, which is equivalent to 3640 weeks. SD rats have an estimated life span of 3 years corresponding to 156 weeks. Ratios of the life spans were calculated. The value obtained was 1:23. Hence, on an average 6 weeks in a rat corresponds to 2.5 years in humans. According to a study by Ahmad et al.,  44% cases developed OSF subsequent to a chewing habit of 2-4 years. In our study, early OSF like changes were seen at 6 weeks which is consistent with findings in the above-mentioned study. Significant increase in fibrosis was evident in the rats following 18-30 weeks of areca nut and pan masala treatment. This amounts to 8-12 years of areca nut/pan masala chewing in humans. Epidemiological studies suggest that prolonged intake of areca nut and pan masala leads to higher grades of OSMF, which is again consistent with our findings. Thus, the model we have attempted to develop closely simulates the development of OSMF in humans.
| Conclusions|| |
In this induced in vivo rat model, we have demonstrated the effects of areca nut extracts and pan masala solutions on the rat buccal mucosa manifesting in the form of atrophic epithelium, connective tissue fibrosis and inflammatory changes. The results from our study reiterate the fact that areca nut and pan masala can have deleterious effects on the oral mucosa contributing to the development of OSF. Furthermore, the destructive potential of areca nut extract appears to be higher than that of the pan masala solution, which is reflected by the capacity of the former to induce more fibrotic changes in a shorter time span as compared to its commercial analogues.
The relevance of a particular animal model to a human disease rests on its ability to parallel the biological changes that characterize the disease in humans.  Although it is unlikely that animal models fully mimic the human disease, our findings bear a close resemblance to the characteristic histological features of OSMF as seen in humans.
The present study showed promising results using a limited number of SD rats. It would be interesting to expand the study model with larger groups to further substantiate the present observations. In addition, the mode of application employed by us (injections) is not the same in usage of areca nut and its products by humans. The evaluation is based on the biological premise of assimilation of the ingredients in the oral mucosa. The absorption of the contents of the "chew" initiates the tissue changes similar to the deposition of the contents into the buccal mucosa by injections in rats.
The results from our study also suggest that SD rats are a sustainable and reproducible model for OSF as appreciable results are obtained within reasonable periods. Moreover, these animals are easy to handle, inexpensive, and universally available at all animal laboratories. Thus, SD rats serve as an excellent model for the extensive research work that needs to be done with regard to OSMF in the absence of sufficient clinical material. In the present scenario with patients being hesitant to undergo biopsy procedures and clinicians becoming increasingly reluctant to take biopsies in cases of OSMF, only animal models can help researchers excogitate the complex alterations occurring at a histopathological and molecular level, at various stages of the disease. This in turn will help in evolving appropriate therapeutic interventions for the patients.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]