Our study was aimed to investigate the efficacy of ANE-induced OSMF animal model administered through previously reported routes of administration (intrabuccally and intraorally). Furthermore, an evaluation of the best possible method of administration that could closely simulate the occurrence of OSMF in humans was performed. Increasing cases of OSMF validates treatment strategy for assessing the safety and efficacy of the same. The very cause for OSMF, areca nut usage, has been shown in the present study to establish an animal model. Areca nut contains alkaloids like arecoline, preceding and quvacine, and other components like flavonoids and copper. These are responsible for disturbance in the extracellular matrix's normal homeostasis, mainly the collagen synthesis and degradation pathways, which terminally lead to OSMF . Over the past decade, extensive research has been carried out on establishing an animal model for OSMF. Das et al., Raghavendra et al., Chiang et al., and Maria et al. used an intra-buccal injection of ANE as a method of administration, while Perera et al. and Bo et al. used topical application of ANE as a method of administration. With ANE as a causative factor, a dose-dependent relationship naturally exists in vivo that has been mimicked for the frequency and duration of exposure and development of a model.
This study simulated the human oral cavity environment with the mice after administering ANE in droplet form. The present study evaluated one conventional method (intrabuccal injections) and one non-conventional method (intraoral dosing) of ANE administration in animals, unlike other studies where only a single mode of administration was used. ANE through the intraoral dose method has been proved superior to the intra-buccal injection technique. We tried to establish an early model through the intra-buccal injection; it is an invasive method that can induce OSMF quickly but does not simulate the exact pathway of disease occurrence in humans [5,10,11,13,20]. Other drawbacks include less surface area for deposition of substance is available which hinders the absorption of ANE and causes discomfort to the animal, which further gains less co-operation from the animal.
Moreover, the repeated injections cause unnecessary inflammation, edema, and hyperemia, interfering with the usual pathway for OSMF establishment. Apart from the injection method of ANE administration, various methods have been used by authors like feeding with diet , topical application over the buccal mucosa , fed through drinking water . All those methods were successful in the model establishment of OSMF, but none of them could simulate the absorption method in humans on the consumption of areca nut-containing products. ANE administration through water or feed can ensure the intake of areca nut, but the absorption through oral mucosa is minimal and thus requires more time to establish the disease.
The intraoral method is easy to perform, without much discomfort to the animals, making it user-friendly and animal ethics committee friendly, allowing us to use more animals. Last but not least, the benefit was derived in a short period; through daily administration. A single dose daily was sufficient to produce results compared to the twice or thrice daily dosage performed earlier [9,14]. The histopathological analysis confirmed the diagnosis of OSMF, which is characterized by atrophic/ hyperplastic epithelium, increased fibrillar collagen in the connective tissue, and decreased vascularity in the subepithelial connective tissue increased chronic inflammatory cell infiltration . OSMF is principally diagnosed through histopathological analysis; thus, different staining procedures are enough to address the establishment of disease microscopically [5,20,21].
Currently, no definite treatment modality is available for the cure or at least reversal of the OSMF. Therefore, developing a definite and pragmatic animal model at the earliest using the least invasive technique that could be employed for drug screening is of prime importance. The present study revealed that ANE administration through the intraoral route showed noticeable histopathological features of OSMF and relatively higher fibrosis than the ANE administration through the intrabuccal route.