Bridging the Research Gap: Establishing a Rabbit Model for Perianal Fistula (Nassor-E Maqad) Study

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Patil This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4144162/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fistula-in-ano , termed ‘ Nassor-e-Maqad’ in traditional Arabic medicine, presents a challenging perianal pathology necessitating a robust experimental framework for comprehensive inquiry. This investigation endeavors to establish an induced Fistula-in-ano model in New Zealand White (NZW) Rabbits (n=12) utilizing a protocol involving surgical suturing steel wire implantation for durations of 20 days (Group ‘A’) and 40 days (Group ‘B'). The principal aim of this research is to develop an induced Fistula-in-ano model in NZW rabbits, with the objective of enhancing comprehension of perianal disease pathogenesis and exploring novel therapeutic modalities. Given the absence of universally efficacious and complication-free treatments, the development of innovative therapeutic approaches is imperative. Hence, the creation of an optimal animal model capable of simulating persistent anal fistulas with lesions resembling clinical manifestations is vital for preclinical investigations in this domain. Upon imaging (X-ray and perianal ultrasonography) the patency of fistula was evaluated whereas histological evaluation exhibited that animals in Group ‘A’ (n=06) fistula has openings with modest epithelialization subsequent to the removal of the surgical steel suture ring, along with favorable diagnostic outcomes, 20 days post-surgery. Conversely, animals in Group ‘B’ (n=6), upon steel wire removal after 40 days, displayed notable changes in epithelization, indicative of further progression in the induced fistula model. In summary, this study successfully establishes a preclinical rabbit model of perianal fistula devoid of chemical agents, relying solely on surgical induction via surgical suturing steel wire (SSSW). The considerable anatomical and histological parallels between rabbit and human perianal pathophysiology underscore the suitability of rabbits as an experimental model for advancing therapeutic strategies in proctology. Animal Science Gastrointestinal Surgery Induced Fistula-in-ano Nassor-e-Maqad Rabbit Model Perianal Fistula Experimental Study Figures Figure 1 Figure 2 Figure 3 Introduction A. Overview of Perianal Fistulas Fistula-in-ano , a pathological condition characterized by an abnormal passage connecting the anal canal to the perianal skin, presents a significant clinical challenge. While various factors can contribute to the development of anal fistulas, cryptoglandular infection is commonly implicated, particularly among patients lacking comorbidities like anorectal abscesses[ 1 ]. A cohort study has revealed higher prevalence of anal fistulas among males under 60 years of age, predominantly with a disease duration of less than a year. Especially among men, one out of every five patients with an anorectal abscess are prone to developing a fistula. Fistula formation showed strong association with inflammatory bowel disease [ 2 ]. Surgical interventions such as fistulotomy and fistulectomy were commonly employed, with notable comorbidities including Crohn’s disease, systemic hypertension, and diabetes mellitus [ 3 ]. Incidence rates varied by gender, with males exhibiting a higher rate compared to females[ 4 ]. In the pursuit of comprehensively understanding the epidemiology of perianal fistulas, a condition marked by the formation of abnormal connections in the vicinity of the anus, researchers have meticulously analyzed data from 26 distinct studies. Among these, 16 studies furnished adequate data for estimating prevalence, revealing a noteworthy prevalence rate of 1.69 per 0.1 million individuals within the general population. Above all, cryptoglandular infection and Crohn’s disease proved to be the most important etiological factors with prevalence rates of 0.86 and 0.76 per 0.1 million inhabitants respectively [ 5 ]. Despite surgical interventions, anal fistulas often recur, emphasizing the complexity of managing this condition and the need for a multidisciplinary treatment approach[ 6 ]. B. Importance of Animal Models in Medical Research The surgical induction of the Fistula-in-ano model involves a meticulously orchestrated series of procedures[ 7 – 11 ], including anesthesia administration, aseptic preparation, creation of a perianal skin incision, mucosal dissection, and placement of a foreign body to induce a persistent fistula tract. Rigorous postoperative care is required to prevent secondary infections and ensure the well-being of the animals throughout the study period[ 10 , 12 ]. Macroscopic and histological examinations of rabbit anatomy exhibited resemblances to human anatomy, particularly in the structure and composition of the anal canal[ 13 , 14 ]. The NZW rabbits were selected as experimental models due to their anatomical similarity, ease of handling, and availability. These attributes render them a suitable choice for establishing an experimental model to investigate perianal fistulas, thereby facilitating the development of novel treatment modalities[ 15 ]. C. Current Limitations in Perianal Fistula Research The disadvantage of the existing animal models is that the repeated handling and weekly interventions on the animal cause more stress and possible deviations, as the application of chemicals and repeated anesthesia during restraining of the rabbit is difficult. The extant models concerning perianal fistula have been established through the implantation of alternative foreign materials over a duration of 30 days. Nonetheless, the resultant fistulae exhibit constrained epithelization, frequently culminating in premature self-resolution and incomplete manifestation of comprehensive clinical features, such as epithelization, granulation, and fibrous formation within the fistulous tract. D. Rationale for Establishing a Rabbit Model Overall, the establishment of the rabbit Fistula-in-ano model offers a promising platform for further research into anal fistula pathophysiology and treatment strategies. By closely mimicking human fistulas and overcoming limitations observed in other animal models, this experimental approach holds considerable potential for advancing therapeutic interventions in the management of anal fistulas. Leveraging the anatomical and physiological similarities between rabbits and humans, the surgical procedure meticulously replicates key characteristics of human perianal fistulas. The successful development of this rabbit model lays a crucial groundwork for future investigations into the pathophysiology and potential therapies for perianal fistulas. Materials and Methods A. Selection of New Zealand White Rabbits The research protocol obtained ethical clearance from the Institutional Ethical Committee of Government Nezamia Tibbi College, Hyderabad, affiliated with Kaloji Narayana University of Health Sciences, Warangal, Telangana State, under Registration No. 21315221008D. The investigation was carried out at the Rabbit Research Centre within the Department of Animal Genetics and Breeding, College of Veterinary Science, Rajendranagar, Hyderabad, Telangana State. Two distinct groups, designated as "A & B," each group comprising 6 male NZW rabbits with an average weight ranging between 1.5-2kg, were systematically chosen and allocated numerical identifiers following precise weighing procedures. These rabbits were accommodated in a controlled environment devoid of pathogens, featuring hygienic surroundings, appropriate lighting, and ventilation facilities. Indoor conditions were regulated to maintain a temperature range of 24 to 28 ℃, alongside a relative humidity level between 50–70%, ensuring exposure to 12 hours of light per day. Formulaic feeding and unhindered access to water were provided to the rabbits on the day preceding and the day of the surgical intervention. B. Induction of Fistula-in-ano ( Nassor-e-Maqad ) The enclosures housing the rabbits were subjected to regular disinfection and individual maintenance protocols, ensuring provision of ample water and food resources. A mandatory 7-day acclimatization period within the enclosures preceded the commencement of experimental procedures. The weights of the rabbits were meticulously documented on the day of surgical intervention and subsequently at 7-day intervals, totaling six distinct time-points. The surgical induction protocol adhered to precise procedural guidelines, encompassing the administration of general anesthesia, aseptic preparation, identification of anal structures, delineation of the surgical site, and insertion of SSSW sized at 18G through the anal sphincter musculature to establish a persistent fistula tract (Fig. 1). Particular emphasis was placed on postoperative care measures aimed at minimizing the risk of secondary infections and ensuring the welfare of the experimental subjects. Elizabeth collar was applied to all animals post-operatively to avoid self-mutilation until removal of SSSW. An anal fistula was surgically induced in 12 rabbits, with radiographic imaging conducted on post-operative days 1st to confirm the accurate placement of the surgical steel wire (see Fig. 2), in the absence of chemical agents. Evaluation of the fistula formation and its progression was performed on the 20th and 40th post-induction days, followed by further assessment via radiography (fistulogram) (see Fig. 3 and Fig. 4) and perianal sonography (PUS) examination (see Fig. 5 and Fig. 6). C. Surgical Procedures and Techniques The experimental paradigm was instituted under the condition of general anesthesia via the implantation of SSSW to emulate anal fistulas in a rabbit model. A period of one week was designated for acclimatization of the experimental subjects prior to the commencement of the procedure. Following this, the rabbits were placed in a supine position and their extremities were appropriately immobilized. Prior to the initiation of the operative protocol, the experimental cohort was stratified into intervention groups denoted as A and B. The induction of perianal fistulas was achieved through a minor surgical intervention conducted under the influence of general anesthesia. Preparation for the induction of perianal fistula involved initial measures of depilation of perianal hair and subsequent disinfection of the region using a povidone iodine solution. Following this, xylocaine 2% jelly mucilage was applied topically. The surgical procedure for the creation of an anal fistula commenced with the clamping of the designated area, augmented by the use of a retractor to facilitate the insertion of a surgical wire through the anal musculature. Specific target sites on the external surface of the anal canal were delineated, situated approximately 1.5 cm from the anal verge at the 5 and 7 o'clock positions. Subsequently, a SSSW No. 18 was punctured into the identified area, and tissue surrounding the puncture site was excised from the anal canal. In cohorts designated as Group ‘A’ (comprising rabbits subjected to a 20-day induction period) and Group ‘B’ (comprising rabbits subjected to a 40-day induction period), the experimental procedure involved the insertion of a surgical wire into the anal canal at either the 5 o'clock or 7 o'clock position, each subgroup consisting of three rabbits. This subgroup classification was made in order to avoid possible distortions due to the choice of the location of the fistula site. The internal termination of the surgical wire within the anal canal was firmly secured using a needle holder forceps and subsequently extracted, with both extremities of the wire being loosely twisted to mitigate the likelihood of post-procedural removal by the animals. D. Monitoring and Care of Experimental Animals Postoperative management encompassed the application of an antiseptic solution to cleanse the procedural site, followed by individual housing of the rabbits for close monitoring and observation. Throughout the duration of the study, vital signs of the rabbits were consistently monitored through cage-site observation. Additionally, the rabbits were provided with unrestricted access to food and water. Regular daily assessments were conducted to ensure their well-being, with provisions made to replenish food and water as required. E. Duration of Study and Follow-Up Protocols Following a period of 20 days, a subgroup consisting of six rabbits from Group-A underwent perianal examination (see Fig. 7) to determine the Perianal Disease Activity Index (PDAI) both prior to and subsequent to the removal of the surgical wire. Following this assessment, these subjects underwent X-ray and perianal ultrasonography (PUS) procedures to evaluate the presence of fistulae before being euthanized. Tissue specimens were then collected for histopathological examination, with corresponding histological scores documented. The same investigative procedures were subsequently applied to the rabbits in Group ‘B’ after a duration of 40 days (see Fig. 8). Model assessment The process of clinical assessment involves careful observation and palpation techniques aimed at evaluating various parameters pertinent to perianal health in experimental animals. The observational phase entails a thorough scrutiny of daily behavioral patterns, food and water intake, fluctuations in body weight, and a detailed examination of the perianal region. Palpation, conversely, involves a comprehensive assessment protocol, encompassing the evaluation of discharge, pain thresholds, limitations in activities, constraints on sexual function, perianal disease classification, and the degree of tissue induration. Furthermore, the Perianal Disease Activity Index (PDAI) score is utilized to authenticate the presence of fistula formation, with regular surveillance of animal mortality. Morphological changes in the perianal region subsequent to SSSW induction are meticulously documented and graded, with subsequent assessments conducted at predetermined intervals. Animals in Group ‘A’ undergo examination 20 days post-SSSW insertion to confirm fistula formation, followed by comprehensive perianal evaluation and removal of SSSW prior to euthanasia. Conversely, animals in Group ‘B’ undergo periodic examinations to assess fistula formation, with specialized imaging modalities such as X-ray Fistulogram and perianal ultrasound (PUS) conducted by proficient radiologists and sonologists. The presence of epithelialization at external openings and fistula tracts is methodically recorded both before and after SSSW removal, ensuring a thorough assessment of Fistula-in-ano development in the rabbit model (see Fig. 7 and Fig. 8). Perianal Disease Activity Index (PDAI): The Perianal Disease Activity Index (PDAI) constitutes a pivotal tool for assess the activity of fistulas in perianal disease, serving as a comprehensive assessment framework. It encompasses the evaluation of five fundamental components: fistula discharge, pain and functional impairment, constraints on sexual activity, type of perianal disease, and extent of induration, delineated in Table 1 to Table 11. A PDAI score surpassing 4 indicates active fistula drainage or local inflammation, with an appreciable accuracy rate of 87%[ 16 ]. This scoring system has undergone rigorous validation in numerous clinical trials, thus fortifying its reliability and applicability in clinical settings. A more straightforward approach to gauge fistula activity involves the "fistula drainage assessment," which relies on the examiner's direct observation of pus discharge upon digital compression. Clinical improvement or response is conventionally signified by a reduction of 50% or more in the number of draining fistulas over two consecutive examinations [ 17 ], while remission is affirmed in the absence of draining fistulas over two consecutive examinations. It is pertinent to note that external fistula openings may undergo healing while inflammatory changes persist within the fistula tracts, necessitating supplementary modalities for comprehensive monitoring. In this context, the utilization of Magnetic Resonance Imaging (MRI) or endoanal ultrasound is advocated for monitoring the progression of perianal fistulas [ 18 ]. A significant advancement in this domain was introduced by van Assche in 2003, who proposed an MRI-based scoring system. Despite its clinical validation, this scoring system demonstrates a modest correlation with the PDAI, indicating prospects for further refinement and optimization in assessing perianal disease activity. Histological examination: Histological analysis unveiled conspicuous epithelialization and granulation tissue formation, notably concentrated in the outer tract region. Noteworthy was the utilization of SSSW as an inflammatory agent, which demonstrated resilience and high tolerability in the rabbit subjects, manifesting no observable alterations throughout a 40-day observation period. However, the challenge in achieving complete epithelialization underscores the intricate progression of fistulas and its potential ramifications for treatment efficacy. Histological assessment revealed that Group ‘A’ (20 days) showed a small fistula tract with irregular patency (see Fig. 9), while Group ‘B’ (40 days) showed uniform patency and a larger area of the fistula tract (see Fig. 10). The tissue damage index (TDI), employing a modified version of the grading system outlined [ 19 ], was employed for histological evaluation, as delineated in Table 3. The histological diagnosis of fistulae depended on specific criteria, including the identification of the internal opening within the rectal mucosa and the external opening on the perianal skin. Histological indicators of proctitis such as neutrophils, B and T lymphocytes and macrophages were also considered. The characterization of the fistulas is based on epithelialization, fibrillation and inflammation. Following the formation of anal fistulas and subsequent cessation of SSSW application, experimental rabbit subjects in each group underwent PUS and X-ray Fistulogram for initial assessment. This encompassed a comprehensive examination of the inner and outer orifices, trajectory of the fistula tract, and surrounding mucosal inflammation. Follow-up PUS and X-ray Fistulogram assessments were performed on the 20th day post-operatively for group "A" and on the 40th day for group "B," respectively. Results The successful induction of Fistula-in-ano in rabbits was assessed via surgical intervention, with subsequent evaluation conducted through clinical examination. Initial observations revealed varying levels of daily activity within both experimental groups, denoted as Groups A and B. Common symptomatic presentations among rabbits in both cohorts encompassed lethargy, sluggishness, and intermittent irritability. A discernible decline in body weight was documented in select specimens across both groups over the study duration. However, by the fourth day post-surgery, activity levels among the rabbits reverted to baseline, accompanied by a normalization of food and water intake. Subsequent to this initial recovery phase, a gradual recuperation in body weight was observed, indicative of conventional perianal healing processes. Notably, rectal prolapse incidents remained absent throughout the entirety of the investigative period. The Mann-Whitney U test results showed that there is no significant difference in scores between Group ‘A’ and Group ‘B’ at any of the three time points (01st, 10th and 20th day) (p > 0.05). However, when 20 days lesions and 40 days lesions were graded and compared using non-parametric test, it indicates that there is a statistically significant difference in the grading of Perianal Fistula development between rabbits in Group ‘A’ (20 days induction period) and Group ‘B’ (40 days induction period). The detailed score card and analysis is available in Annexure 1. Following surgical intervention, the perianal regions of experimental animals encompassing both groups A and B underwent assessment for Perianal Disease Activity Index (PDAI) scores one day post-surgery, accompanied by X-ray examination to validate the successful induction of the SSSW. On the tenth day post-surgery, subjects from both cohorts were subjected to PDAI evaluation, with subsequent reassessment on the twentieth day. In Group ‘A’, removal of the SSSW from the perianal area of all subjects occurred after the twentieth day, followed by the confirmation of Fistula-in-ano formation and scoring utilizing the PDAI scale. Post-SSSW extraction observation delineated the presence of two distinct openings, internal and external, characterized by evident epithelialization. Manual examination revealed palpable induration, discharge, and tenderness. Thereafter, subjects from Group ‘A’ underwent PUS and X-Ray Fistulogram to ascertain fistula formation prior to euthanasia for histological scrutiny. The grading for PDAI covered fistula discharge (see Fig. 1 and Table 2), pain and functional impairment/restriction of activities (see Table 3 and Table 4), constraints on sexual activity (see Table 5 and Table 6), type of perianal disease (see Table 7 and Table 8), and extent of induration (see Table 9 and Table 10). In contrast to the experimental procedures conducted on six rabbits in Group ‘A’, an identical protocol was executed on six rabbits from Group ‘B’, respectively, on the 30th and 40th days following the implantation of SSSW. Subsequent to the surgically induced Fistula-in-ano in both experimental cohorts, scores were meticulously ascertained and subjected to comparative analysis. The statistical scrutiny divulged markedly elevated scores within Group ‘B’ in relation to Group ‘A’, albeit with the exception of the Perianal Disease Activity Index (PDAI) score (P < 0.05). Furthermore, adjunctive diagnostic modalities, namely PUS and X-ray Fistulogram, were deployed in both experimental cohorts to gauge the extent of fistula formation. It is noteworthy that rabbits allocated to Group ‘B’ exhibited a more conspicuous manifestation of fistula formation, characterized by discernible epithelialization, granulation, and fibrous tissue proliferation forty days post-induction, in contrast to the comparatively subdued outcomes observed in Group ‘A’. These empirical observations collectively infer the superiority of the employed modeling methodology involving SSSW No. 18G in inducing Fistula-in-ano within Group ‘B’ in contrast to the methodological approach adopted in Group ‘A’. Histological examination was conducted subsequent to the conclusion of the experimental phase, wherein animals were humanely euthanized via thiopentone overdose, and tissue specimens were procured from the perianal region for analysis. Standard hematoxylin and eosin (HE) staining protocols were employed to evaluate the extent of inflammation and other pathological alterations associated with Fistula-in-ano induction. Statistical analysis of the collected data was performed using SPSS 19.0 software, with results presented as mean values accompanied by standard deviations. A significance threshold of P < 0.05 was applied to ascertain statistical significance. Histopathological assessment of tissue samples from animals in Group ‘A’ exhibited pronounced inflammatory manifestations within the fistulous tract, typified by abscess formation, necrotic tissue accumulation, and infiltration of immune cells. Comparative analysis revealed notably elevated pathology scores in Group ‘B’ animals compared to those in Group ‘A’, characterized by a conspicuous presence of the fistulous tract and marked squamous cell epithelization, indicative of a more advanced pathological state. The histological images were analyzed for manual visual assessment of ulcers, lymphocytes, neutrophils, fibrosis, recessions and oedema. Blind analysis was performed by an expert on these slides. The ulcer was observed in both groups (20th and 40th day), but it was less pronounced in Group ‘B’, which could be due to recovery (granulation). The inflammatory cells (neutrophils and lymphocytes) and oedema were present in both groups without major differences. The chronic inflammatory response (fibrosis and recession) was much more pronounced in Group ‘B’ compared to Group ‘A’. The classification can be seen in Table 12. Discussion Perianal fistulas epitomize aberrant tracts or passages that develop in the perianal region, typically stemming from anal gland infection or inflammation. These pathological formations often manifest symptoms such as pain, swelling, and discharge around the anal area. Diagnosis commonly involves clinical evaluation by healthcare practitioners supplemented by imaging modalities such as MRI or ultrasound for comprehensive assessment. Treatment strategies vary based on severity and may encompass antibiotic therapy to address infection, coupled with surgical intervention to excise the fistulous tract and drain associated abscesses. In select cases, pharmacological agents, particularly immunosuppressants, may be prescribed, especially when underlying conditions like Crohn's disease are implicated. Despite therapeutic endeavors, perianal fistulas pose challenges due to their propensity for recurrence and potential complications such as chronic pain and fecal incontinence. Consequently, ongoing management requires vigilance, often necessitating long-term surveillance and continued medical intervention to optimize patient outcomes and mitigate recurrence rates. The management of Fistula-in-ano remains a persistent challenge for clinicians and surgeons alike, characterized by complexities in decision-making regarding surgical interventions, particularly with the advent of novel techniques in recent years [ 20 , 21 ]. Despite the efforts of experienced practitioners, achieving a consensus on optimal surgical approaches for Fistula-in-ano of cryptoglandular origin has proven elusive[ 22 ]. This discourse aims to explore the diverse trajectories of Fistula-in-ano , delineating their respective advantages and drawbacks. The rabbit emerges as a compelling choice for experimental and investigative endeavors in proctology, given its striking anatomical and histological resemblance to humans, as well as its manageable size and ease of manipulation. Our research endeavors to minimize animal distress while maximizing research outcomes through innovative methodologies. Notably, we present a novel approach to induce Fistula-in-ano in rabbits using solely SSSW No. 18G under general anesthesia, eschewing the use of chemical agents. Presently, no universally optimal curative treatment exists for this condition. In comparison to smaller animal models such as rats, rabbits, with their analogous anatomy to humans, offer a more suitable platform for studying Fistula-in-ano. Our investigation furnishes compelling morphological and histological evidence corroborating the resemblance between rabbit anatomy and that of humans, particularly concerning perineal and sphincter musculatures. The induction of rectal histological inflammation with perianal trans-sphincteric fistula formation was carefully observed through PUS and X-Ray Fistulogram [ 23 ]. Diagnosis primarily relied on PDAIS and histology, complemented by diagnostic modalities such as PUS and X-Ray Fistulogram. In this animal model, the induction of SSSW stimulates inflammation in the perianal region, initiating the wound healing cascade. Our approach, involved one time fistula induction using SSSW under general anesthesia, mimics human fistula recurrence while prolonging inflammation, thereby eliciting a robust inflammatory response. Notably, persistent inflammation induction led to improved disease scores in Group ‘B’, underscoring the significance of sustained stimulus for optimal fistula formation. Our findings demonstrate that a preclinical model of perianal fistula can be effectively established in rabbits with prolonged intact SSSW induction. Histopathological examination revealed features consistent with human perianal fistula, albeit further criteria validation is warranted. Despite the promising outcomes observed in our rabbit model, it is imperative to acknowledge the inherent limitations of this study. Unlike previous studies utilizing canine and porcine models, our approach showcased robust epithelialization and tissue formation post-fistula induction, suggesting the potential utility of the rabbit model in advancing our understanding and management of Fistula-in-ano. Conclusion In summary, this study conceals the development of a direct preclinical animal model that simulates perianal fistula formation in rabbits by using an extended technique to induce perianal fistula by prolonged (40 days) implantation of SSSW. This model is capable of mimicking the pathological condition in humans, whereby the induced fistula lesions can be assessed by PDAIS, histological analysis and additional diagnostic procedures such as PUS and X-ray examinations of the perianal region. The comprehensive evaluation carried out here should enrich research into innovative therapeutic strategies for the treatment of perianal fistula diseases. References Garcia-Aguilar, J., et al., Anal fistula surgery: factors associated with recurrence and incontinence. 1996. 39 : p. 723-729. Sanchez-Haro, E., et al., Clinical characterization of patients with anal fistula during follow-up of anorectal abscess: a large population-based study. 2023. 27 (10): p. 897-907. Krishnakanth, M., Evaluation of Fistulectomy and Fistulotomy in the Management of Low Anal Fistula . 2018, Rajiv Gandhi University of Health Sciences (India). Hansdah, S. and T.J.A.o.t.R.S.f.C.B. Baxla, The Incidence of low Fistula in Ano and RESULTS of Fistulectomy. 2021. 25 (6): p. 6794-6797. Garcia-Olmo, D., et al., Prevalence of anal fistulas in Europe: systematic literature reviews and population-based database analysis. 2019. 36 (12): p. 3503-3518. Vogel, J.D., et al., Clinical practice guideline for the management of anorectal abscess, fistula-in-ano, and rectovaginal fistula. 2016. 59 (12): p. 1117-1133. Lu, S., et al., Evaluation of animal models of Crohn's disease with anal fistula. 2021. 22 (3): p. 1-7. Rafati, M., et al., Human amniotic membrane effect on perianal fistula healing in rabbits: an experimental study. 2018. 43 (5): p. 473. Wang, T., et al., Injectable and self-healing microgels repairing infected irregular tubular fistula in anal and muscle. 2022. 441 : p. 135948. Singer, M., et al., Treatment of fistulas-in-ano with fibrin sealant in combination with intra-adhesive antibiotics and/or surgical closure of the internal fistula opening. 2005. 48 (4): p. 799-808. Flacs, M., et al., Preclinical model of perianal fistulizing Crohn’s disease. 2020. 26 (5): p. 687-696. Singh, A., et al., Management of perianal fistulizing Crohn’s disease. 2023: p. izad195. Kural, T., Microscopic Structure of Intestinal Anastomoses and the Use of Animal Models in Experimental Intestinal Surgery. 2023. Benlice, C., et al., Fistula tract curettage and the use of biological dermal plugs improve high transsphincteric fistula healing in an animal model. 2016. 31 : p. 291-299. De la Portilla, F., et al., The rabbit as an animal model for proctology research: anatomical and histological description. 2011. 24 (3): p. 134-137. Losco, A., et al., Assessing the activity of perianal Crohn's disease: comparison of clinical indices and computer-assisted anal ultrasound. Inflamm Bowel Dis, 2009. 15 (5): p. 742-9. Gecse, K.B., et al., Results of the Fifth Scientific Workshop of the ECCO [II]: Clinical Aspects of Perianal Fistulising Crohn’s Disease—the Unmet Needs. Journal of Crohn's and Colitis, 2016. 10 (7): p. 758-765. Akhoundi, N., et al., Comparison of MRI and Endoanal Ultrasound in Assessing Intersphincteric, Transsphincteric, and Suprasphincteric Perianal Fistula. J Ultrasound Med, 2023. 42 (9): p. 2057-2064. MacPherson, B.R. and C.J. Pfeiffer, Experimental production of diffuse colitis in rats. Digestion, 1978. 17 (2): p. 135-50. Kontovounisios, C., et al., Adoption and success rates of perineal procedures for fistula‐in‐ano: a systematic review. 2016. 18 (5): p. 441-458. Deeba, S., et al., Fistula-in-ano: advances in treatment. 2008. 196 (1): p. 95-99. Ji, L., et al., Advances in the treatment of anal fistula: a mini-review of recent five-year clinical studies. 2021. 7 : p. 586891. Shahzad, M., et al., Effectiveness of radiological imaging techniques (x-rays, mdct, and mri) for diagnosis of pelvic fistula: A systematic review. 2021. 2021 (1). Tables Tables 1 to 12 are available in the Supplementary Files section. Annexure Annexure 1 is not available with this version. Additional Declarations The authors declare no competing interests. Supplementary Files Table12.jpg Table 1-2 Table34.jpg Table 3-4 Table56.jpg Table 5-6 Table78.jpg Table 7-8 Table910.jpg Table 9-10 Table1112.jpg Table 11-12 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4144162","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282316803,"identity":"5939778f-7671-4947-b8d6-1413cc11bd77","order_by":0,"name":"Syed Abdul Shakoor Bukhari","email":"data:image/png;base64,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","orcid":"","institution":"PG Scholar (Ilmul-Advia) Pharmacology, Government Nizamia Tibbia College, Charminar, Hyderabad T.S, Medical Officer, Department of AYUSH, J \u0026 K, India","correspondingAuthor":true,"prefix":"","firstName":"Syed","middleName":"Abdul Shakoor","lastName":"Bukhari","suffix":""},{"id":282316804,"identity":"236e9051-ff13-4a0c-a63d-a94ad69130d2","order_by":1,"name":"Zaibunnisa Begum","email":"","orcid":"","institution":"Head \u0026 Professor, PG Department of Pharmacology GNTC Hyderabad T.S","correspondingAuthor":false,"prefix":"","firstName":"Zaibunnisa","middleName":"","lastName":"Begum","suffix":""},{"id":282316805,"identity":"d4e87e32-d2f6-473d-9283-dd0692678093","order_by":2,"name":"Pradeep B. Patil","email":"","orcid":"","institution":"MVSc, PhD, PDF (Tissue Engineering) Scientist D, ICMR-NIN, Jamai-Osmania PO, Hyderabad -007","correspondingAuthor":false,"prefix":"","firstName":"Pradeep","middleName":"B.","lastName":"Patil","suffix":""}],"badges":[],"createdAt":"2024-03-21 14:16:59","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4144162/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4144162/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53500442,"identity":"0f77fc8e-4ae3-4737-adb8-7e69055f004a","added_by":"auto","created_at":"2024-03-26 18:11:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1750233,"visible":true,"origin":"","legend":"\u003cp\u003eFig 1-4 See image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/91f93fd6b027f1920df20559.jpg"},{"id":53500446,"identity":"223b0213-b3e0-434a-8cc3-4bfe6da100ec","added_by":"auto","created_at":"2024-03-26 18:11:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1529557,"visible":true,"origin":"","legend":"\u003cp\u003eFig 5-8 See image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure58reserachSquarepage0001.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/0b3abb01e9333a410de5fbed.jpg"},{"id":53500448,"identity":"fcb1e4b8-434a-4cd6-aa1c-a2777c2ce542","added_by":"auto","created_at":"2024-03-26 18:11:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2482066,"visible":true,"origin":"","legend":"\u003cp\u003eFig 9-10 See image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig910.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/f044c572cf9bb6a764b9c21e.jpg"},{"id":53501867,"identity":"4368688e-a560-47ac-bc23-d661a8881a9f","added_by":"auto","created_at":"2024-03-26 18:27:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":790838,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/60202da7-4582-46ee-81f1-e7e597d63673.pdf"},{"id":53500443,"identity":"f4a63dd0-f7c4-49e7-97f6-be0689b645b4","added_by":"auto","created_at":"2024-03-26 18:11:47","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":627337,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1-2\u003c/p\u003e","description":"","filename":"Table12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/c9d59daf1f3c0bfc3a753dfb.jpg"},{"id":53500447,"identity":"78cfa87b-369f-478e-a98d-41edc6d6d2b3","added_by":"auto","created_at":"2024-03-26 18:11:48","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":463751,"visible":true,"origin":"","legend":"\u003cp\u003eTable 3-4\u003c/p\u003e","description":"","filename":"Table34.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/4c8a530d02ae410a5a6c696b.jpg"},{"id":53500451,"identity":"7a1a258b-e55f-4957-a09b-62a413fdff17","added_by":"auto","created_at":"2024-03-26 18:11:48","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":496419,"visible":true,"origin":"","legend":"\u003cp\u003eTable 5-6\u003c/p\u003e","description":"","filename":"Table56.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/3e180ba2d6beb237f5fed80d.jpg"},{"id":53500449,"identity":"9ec62186-6669-400e-bf45-77cb5b7d09eb","added_by":"auto","created_at":"2024-03-26 18:11:48","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":448221,"visible":true,"origin":"","legend":"\u003cp\u003eTable 7-8\u003c/p\u003e","description":"","filename":"Table78.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/0dd93ff3452064bac745ae3b.jpg"},{"id":53500444,"identity":"cb0e0089-ece4-45eb-903c-2b8f575c55ca","added_by":"auto","created_at":"2024-03-26 18:11:47","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":552588,"visible":true,"origin":"","legend":"\u003cp\u003eTable 9-10\u003c/p\u003e","description":"","filename":"Table910.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/abd10e6cfb2459a38a8359d0.jpg"},{"id":53501452,"identity":"d53db574-2293-4f3c-afed-9d7947230e73","added_by":"auto","created_at":"2024-03-26 18:19:48","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":721479,"visible":true,"origin":"","legend":"\u003cp\u003eTable 11-12\u003c/p\u003e","description":"","filename":"Table1112.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4144162/v1/bedac8e396f00b7cb59a1fc4.jpg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eBridging the Research Gap: Establishing a Rabbit Model for Perianal Fistula (\u003cem\u003eNassor-E Maqad\u003c/em\u003e) Study\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA. Overview of Perianal Fistulas\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFistula-in-ano\u003c/em\u003e, a pathological condition characterized by an abnormal passage connecting the anal canal to the perianal skin, presents a significant clinical challenge. While various factors can contribute to the development of anal fistulas, cryptoglandular infection is commonly implicated, particularly among patients lacking comorbidities like anorectal abscesses[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. A cohort study has revealed higher prevalence of anal fistulas among males under 60 years of age, predominantly with a disease duration of less than a year. Especially among men, one out of every five patients with an anorectal abscess are prone to developing a fistula. Fistula formation showed strong association with inflammatory bowel disease [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Surgical interventions such as fistulotomy and fistulectomy were commonly employed, with notable comorbidities including Crohn\u0026rsquo;s disease, systemic hypertension, and \u003cem\u003ediabetes mellitus\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Incidence rates varied by gender, with males exhibiting a higher rate compared to females[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the pursuit of comprehensively understanding the epidemiology of perianal fistulas, a condition marked by the formation of abnormal connections in the vicinity of the anus, researchers have meticulously analyzed data from 26 distinct studies. Among these, 16 studies furnished adequate data for estimating prevalence, revealing a noteworthy prevalence rate of 1.69 per 0.1\u0026nbsp;million individuals within the general population. Above all, cryptoglandular infection and Crohn\u0026rsquo;s disease proved to be the most important etiological factors with prevalence rates of 0.86 and 0.76 per 0.1\u0026nbsp;million inhabitants respectively [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite surgical interventions, anal fistulas often recur, emphasizing the complexity of managing this condition and the need for a multidisciplinary treatment approach[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eB. Importance of Animal Models in Medical Research\u003c/p\u003e\n\u003cp\u003eThe surgical induction of the \u003cem\u003eFistula-in-ano\u003c/em\u003e model involves a meticulously orchestrated series of procedures[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], including anesthesia administration, aseptic preparation, creation of a perianal skin incision, mucosal dissection, and placement of a foreign body to induce a persistent fistula tract. Rigorous postoperative care is required to prevent secondary infections and ensure the well-being of the animals throughout the study period[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eMacroscopic and histological examinations of rabbit anatomy exhibited resemblances to human anatomy, particularly in the structure and composition of the anal canal[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The NZW rabbits were selected as experimental models due to their anatomical similarity, ease of handling, and availability. These attributes render them a suitable choice for establishing an experimental model to investigate perianal fistulas, thereby facilitating the development of novel treatment modalities[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eC. Current Limitations in Perianal Fistula Research\u003c/p\u003e\n\u003cp\u003eThe disadvantage of the existing animal models is that the repeated handling and weekly interventions on the animal cause more stress and possible deviations, as the application of chemicals and repeated anesthesia during restraining of the rabbit is difficult. The extant models concerning perianal fistula have been established through the implantation of alternative foreign materials over a duration of 30 days. Nonetheless, the resultant fistulae exhibit constrained epithelization, frequently culminating in premature self-resolution and incomplete manifestation of comprehensive clinical features, such as epithelization, granulation, and fibrous formation within the fistulous tract.\u003c/p\u003e\n\u003cp\u003eD. Rationale for Establishing a Rabbit Model\u003c/p\u003e\n\u003cp\u003eOverall, the establishment of the rabbit \u003cem\u003eFistula-in-ano\u003c/em\u003e model offers a promising platform for further research into anal fistula pathophysiology and treatment strategies. By closely mimicking human fistulas and overcoming limitations observed in other animal models, this experimental approach holds considerable potential for advancing therapeutic interventions in the management of anal fistulas. Leveraging the anatomical and physiological similarities between rabbits and humans, the surgical procedure meticulously replicates key characteristics of human perianal fistulas. The successful development of this rabbit model lays a crucial groundwork for future investigations into the pathophysiology and potential therapies for perianal fistulas.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eA. Selection of New Zealand White Rabbits\u003c/p\u003e \u003cp\u003e The research protocol obtained ethical clearance from the Institutional Ethical Committee of Government Nezamia Tibbi College, Hyderabad, affiliated with Kaloji Narayana University of Health Sciences, Warangal, Telangana State, under Registration No. 21315221008D. The investigation was carried out at the Rabbit Research Centre within the Department of Animal Genetics and Breeding, College of Veterinary Science, Rajendranagar, Hyderabad, Telangana State. Two distinct groups, designated as \"A \u0026amp; B,\" each group comprising 6 male NZW rabbits with an average weight ranging between 1.5-2kg, were systematically chosen and allocated numerical identifiers following precise weighing procedures. These rabbits were accommodated in a controlled environment devoid of pathogens, featuring hygienic surroundings, appropriate lighting, and ventilation facilities. Indoor conditions were regulated to maintain a temperature range of 24 to 28 ℃, alongside a relative humidity level between 50\u0026ndash;70%, ensuring exposure to 12 hours of light per day. Formulaic feeding and unhindered access to water were provided to the rabbits on the day preceding and the day of the surgical intervention.\u003c/p\u003e \u003cp\u003eB. Induction of \u003cem\u003eFistula-in-ano\u003c/em\u003e (\u003cem\u003eNassor-e-Maqad\u003c/em\u003e)\u003c/p\u003e \u003cp\u003eThe enclosures housing the rabbits were subjected to regular disinfection and individual maintenance protocols, ensuring provision of ample water and food resources. A mandatory 7-day acclimatization period within the enclosures preceded the commencement of experimental procedures. The weights of the rabbits were meticulously documented on the day of surgical intervention and subsequently at 7-day intervals, totaling six distinct time-points. The surgical induction protocol adhered to precise procedural guidelines, encompassing the administration of general anesthesia, aseptic preparation, identification of anal structures, delineation of the surgical site, and insertion of SSSW sized at 18G through the anal sphincter musculature to establish a persistent fistula tract (Fig.\u0026nbsp;1). Particular emphasis was placed on postoperative care measures aimed at minimizing the risk of secondary infections and ensuring the welfare of the experimental subjects. Elizabeth collar was applied to all animals post-operatively to avoid self-mutilation until removal of SSSW. An anal fistula was surgically induced in 12 rabbits, with radiographic imaging conducted on post-operative days 1st to confirm the accurate placement of the surgical steel wire (see Fig.\u0026nbsp;2), in the absence of chemical agents. Evaluation of the fistula formation and its progression was performed on the 20th and 40th post-induction days, followed by further assessment \u003cem\u003evia\u003c/em\u003e radiography (fistulogram) (see Fig.\u0026nbsp;3 and Fig.\u0026nbsp;4) and perianal sonography (PUS) examination (see Fig.\u0026nbsp;5 and Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eC. Surgical Procedures and Techniques\u003c/p\u003e \u003cp\u003eThe experimental paradigm was instituted under the condition of general anesthesia \u003cem\u003evia\u003c/em\u003e the implantation of SSSW to emulate anal fistulas in a rabbit model. A period of one week was designated for acclimatization of the experimental subjects prior to the commencement of the procedure. Following this, the rabbits were placed in a supine position and their extremities were appropriately immobilized. Prior to the initiation of the operative protocol, the experimental cohort was stratified into intervention groups denoted as A and B. The induction of perianal fistulas was achieved through a minor surgical intervention conducted under the influence of general anesthesia.\u003c/p\u003e \u003cp\u003ePreparation for the induction of perianal fistula involved initial measures of depilation of perianal hair and subsequent disinfection of the region using a povidone iodine solution. Following this, xylocaine 2% jelly mucilage was applied topically. The surgical procedure for the creation of an anal fistula commenced with the clamping of the designated area, augmented by the use of a retractor to facilitate the insertion of a surgical wire through the anal musculature. Specific target sites on the external surface of the anal canal were delineated, situated approximately 1.5 cm from the anal verge at the 5 and 7 o'clock positions. Subsequently, a SSSW No. 18 was punctured into the identified area, and tissue surrounding the puncture site was excised from the anal canal.\u003c/p\u003e \u003cp\u003eIn cohorts designated as Group \u0026lsquo;A\u0026rsquo; (comprising rabbits subjected to a 20-day induction period) and Group \u0026lsquo;B\u0026rsquo; (comprising rabbits subjected to a 40-day induction period), the experimental procedure involved the insertion of a surgical wire into the anal canal at either the 5 o'clock or 7 o'clock position, each subgroup consisting of three rabbits. This subgroup classification was made in order to avoid possible distortions due to the choice of the location of the fistula site. The internal termination of the surgical wire within the anal canal was firmly secured using a needle holder forceps and subsequently extracted, with both extremities of the wire being loosely twisted to mitigate the likelihood of post-procedural removal by the animals.\u003c/p\u003e \u003cp\u003eD. Monitoring and Care of Experimental Animals\u003c/p\u003e \u003cp\u003e Postoperative management encompassed the application of an antiseptic solution to cleanse the procedural site, followed by individual housing of the rabbits for close monitoring and observation. Throughout the duration of the study, vital signs of the rabbits were consistently monitored through cage-site observation. Additionally, the rabbits were provided with unrestricted access to food and water. Regular daily assessments were conducted to ensure their well-being, with provisions made to replenish food and water as required.\u003c/p\u003e \u003cp\u003eE. Duration of Study and Follow-Up Protocols\u003c/p\u003e \u003cp\u003eFollowing a period of 20 days, a subgroup consisting of six rabbits from Group-A underwent perianal examination (see Fig.\u0026nbsp;7) to determine the Perianal Disease Activity Index (PDAI) both prior to and subsequent to the removal of the surgical wire. Following this assessment, these subjects underwent X-ray and perianal ultrasonography (PUS) procedures to evaluate the presence of fistulae before being euthanized. Tissue specimens were then collected for histopathological examination, with corresponding histological scores documented. The same investigative procedures were subsequently applied to the rabbits in Group \u0026lsquo;B\u0026rsquo; after a duration of 40 days (see Fig.\u0026nbsp;8).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eModel assessment\u003c/h2\u003e \u003cp\u003e The process of clinical assessment involves careful observation and palpation techniques aimed at evaluating various parameters pertinent to perianal health in experimental animals. The observational phase entails a thorough scrutiny of daily behavioral patterns, food and water intake, fluctuations in body weight, and a detailed examination of the perianal region. Palpation, conversely, involves a comprehensive assessment protocol, encompassing the evaluation of discharge, pain thresholds, limitations in activities, constraints on sexual function, perianal disease classification, and the degree of tissue induration. Furthermore, the Perianal Disease Activity Index (PDAI) score is utilized to authenticate the presence of fistula formation, with regular surveillance of animal mortality. Morphological changes in the perianal region subsequent to SSSW induction are meticulously documented and graded, with subsequent assessments conducted at predetermined intervals. Animals in Group \u0026lsquo;A\u0026rsquo; undergo examination 20 days post-SSSW insertion to confirm fistula formation, followed by comprehensive perianal evaluation and removal of SSSW prior to euthanasia. Conversely, animals in Group \u0026lsquo;B\u0026rsquo; undergo periodic examinations to assess fistula formation, with specialized imaging modalities such as X-ray Fistulogram and perianal ultrasound (PUS) conducted by proficient radiologists and sonologists. The presence of epithelialization at external openings and fistula tracts is methodically recorded both before and after SSSW removal, ensuring a thorough assessment of \u003cem\u003eFistula-in-ano\u003c/em\u003e development in the rabbit model (see Fig.\u0026nbsp;7 and Fig.\u0026nbsp;8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePerianal Disease Activity Index (PDAI):\u003c/h2\u003e \u003cp\u003eThe Perianal Disease Activity Index (PDAI) constitutes a pivotal tool for assess the activity of fistulas in perianal disease, serving as a comprehensive assessment framework. It encompasses the evaluation of five fundamental components: fistula discharge, pain and functional impairment, constraints on sexual activity, type of perianal disease, and extent of induration, delineated in Table\u0026nbsp;1 to Table\u0026nbsp;11. A PDAI score surpassing 4 indicates active fistula drainage or local inflammation, with an appreciable accuracy rate of 87%[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This scoring system has undergone rigorous validation in numerous clinical trials, thus fortifying its reliability and applicability in clinical settings.\u003c/p\u003e \u003cp\u003eA more straightforward approach to gauge fistula activity involves the \"fistula drainage assessment,\" which relies on the examiner's direct observation of pus discharge upon digital compression. Clinical improvement or response is conventionally signified by a reduction of 50% or more in the number of draining fistulas over two consecutive examinations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], while remission is affirmed in the absence of draining fistulas over two consecutive examinations. It is pertinent to note that external fistula openings may undergo healing while inflammatory changes persist within the fistula tracts, necessitating supplementary modalities for comprehensive monitoring.\u003c/p\u003e \u003cp\u003eIn this context, the utilization of Magnetic Resonance Imaging (MRI) or endoanal ultrasound is advocated for monitoring the progression of perianal fistulas [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A significant advancement in this domain was introduced by van Assche in 2003, who proposed an MRI-based scoring system. Despite its clinical validation, this scoring system demonstrates a modest correlation with the PDAI, indicating prospects for further refinement and optimization in assessing perianal disease activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistological examination:\u003c/h2\u003e \u003cp\u003eHistological analysis unveiled conspicuous epithelialization and granulation tissue formation, notably concentrated in the outer tract region. Noteworthy was the utilization of SSSW as an inflammatory agent, which demonstrated resilience and high tolerability in the rabbit subjects, manifesting no observable alterations throughout a 40-day observation period. However, the challenge in achieving complete epithelialization underscores the intricate progression of fistulas and its potential ramifications for treatment efficacy. Histological assessment revealed that Group \u0026lsquo;A\u0026rsquo; (20 days) showed a small fistula tract with irregular patency (see Fig.\u0026nbsp;9), while Group \u0026lsquo;B\u0026rsquo; (40 days) showed uniform patency and a larger area of the fistula tract (see Fig.\u0026nbsp;10).\u003c/p\u003e \u003cp\u003eThe tissue damage index (TDI), employing a modified version of the grading system outlined [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], was employed for histological evaluation, as delineated in Table\u0026nbsp;3. The histological diagnosis of fistulae depended on specific criteria, including the identification of the internal opening within the rectal mucosa and the external opening on the perianal skin. Histological indicators of proctitis such as neutrophils, B and T lymphocytes and macrophages were also considered. The characterization of the fistulas is based on epithelialization, fibrillation and inflammation.\u003c/p\u003e \u003cp\u003eFollowing the formation of anal fistulas and subsequent cessation of SSSW application, experimental rabbit subjects in each group underwent PUS and X-ray Fistulogram for initial assessment. This encompassed a comprehensive examination of the inner and outer orifices, trajectory of the fistula tract, and surrounding mucosal inflammation. Follow-up PUS and X-ray Fistulogram assessments were performed on the 20th day post-operatively for group \"A\" and on the 40th day for group \"B,\" respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe successful induction of \u003cem\u003eFistula-in-ano\u003c/em\u003e in rabbits was assessed via surgical intervention, with subsequent evaluation conducted through clinical examination. Initial observations revealed varying levels of daily activity within both experimental groups, denoted as Groups A and B. Common symptomatic presentations among rabbits in both cohorts encompassed lethargy, sluggishness, and intermittent irritability. A discernible decline in body weight was documented in select specimens across both groups over the study duration. However, by the fourth day post-surgery, activity levels among the rabbits reverted to baseline, accompanied by a normalization of food and water intake. Subsequent to this initial recovery phase, a gradual recuperation in body weight was observed, indicative of conventional perianal healing processes. Notably, rectal prolapse incidents remained absent throughout the entirety of the investigative period.\u003c/p\u003e \u003cp\u003eThe Mann-Whitney U test results showed that there is no significant difference in scores between Group \u0026lsquo;A\u0026rsquo; and Group \u0026lsquo;B\u0026rsquo; at any of the three time points (01st, 10th and 20th day) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, when 20 days lesions and 40 days lesions were graded and compared using non-parametric test, it indicates that there is a statistically significant difference in the grading of Perianal Fistula development between rabbits in Group \u0026lsquo;A\u0026rsquo; (20 days induction period) and Group \u0026lsquo;B\u0026rsquo; (40 days induction period). The detailed score card and analysis is available in Annexure 1.\u003c/p\u003e \u003cp\u003eFollowing surgical intervention, the perianal regions of experimental animals encompassing both groups A and B underwent assessment for Perianal Disease Activity Index (PDAI) scores one day post-surgery, accompanied by X-ray examination to validate the successful induction of the SSSW. On the tenth day post-surgery, subjects from both cohorts were subjected to PDAI evaluation, with subsequent reassessment on the twentieth day. In Group \u0026lsquo;A\u0026rsquo;, removal of the SSSW from the perianal area of all subjects occurred after the twentieth day, followed by the confirmation of \u003cem\u003eFistula-in-ano\u003c/em\u003e formation and scoring utilizing the PDAI scale. Post-SSSW extraction observation delineated the presence of two distinct openings, internal and external, characterized by evident epithelialization. Manual examination revealed palpable induration, discharge, and tenderness. Thereafter, subjects from Group \u0026lsquo;A\u0026rsquo; underwent PUS and X-Ray Fistulogram to ascertain fistula formation prior to euthanasia for histological scrutiny. The grading for PDAI covered fistula discharge (see Fig.\u0026nbsp;1 and Table\u0026nbsp;2), pain and functional impairment/restriction of activities (see Table\u0026nbsp;3 and Table\u0026nbsp;4), constraints on sexual activity (see Table\u0026nbsp;5 and Table\u0026nbsp;6), type of perianal disease (see Table\u0026nbsp;7 and Table\u0026nbsp;8), and extent of induration (see Table\u0026nbsp;9 and Table\u0026nbsp;10).\u003c/p\u003e \u003cp\u003eIn contrast to the experimental procedures conducted on six rabbits in Group \u0026lsquo;A\u0026rsquo;, an identical protocol was executed on six rabbits from Group \u0026lsquo;B\u0026rsquo;, respectively, on the 30th and 40th days following the implantation of SSSW. Subsequent to the surgically induced \u003cem\u003eFistula-in-ano\u003c/em\u003e in both experimental cohorts, scores were meticulously ascertained and subjected to comparative analysis. The statistical scrutiny divulged markedly elevated scores within Group \u0026lsquo;B\u0026rsquo; in relation to Group \u0026lsquo;A\u0026rsquo;, albeit with the exception of the Perianal Disease Activity Index (PDAI) score (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, adjunctive diagnostic modalities, namely PUS and X-ray Fistulogram, were deployed in both experimental cohorts to gauge the extent of fistula formation.\u003c/p\u003e \u003cp\u003eIt is noteworthy that rabbits allocated to Group \u0026lsquo;B\u0026rsquo; exhibited a more conspicuous manifestation of fistula formation, characterized by discernible epithelialization, granulation, and fibrous tissue proliferation forty days post-induction, in contrast to the comparatively subdued outcomes observed in Group \u0026lsquo;A\u0026rsquo;. These empirical observations collectively infer the superiority of the employed modeling methodology involving SSSW No. 18G in inducing \u003cem\u003eFistula-in-ano\u003c/em\u003e within Group \u0026lsquo;B\u0026rsquo; in contrast to the methodological approach adopted in Group \u0026lsquo;A\u0026rsquo;.\u003c/p\u003e \u003cp\u003eHistological examination was conducted subsequent to the conclusion of the experimental phase, wherein animals were humanely euthanized via thiopentone overdose, and tissue specimens were procured from the perianal region for analysis. Standard hematoxylin and eosin (HE) staining protocols were employed to evaluate the extent of inflammation and other pathological alterations associated with \u003cem\u003eFistula-in-ano\u003c/em\u003e induction. Statistical analysis of the collected data was performed using SPSS 19.0 software, with results presented as mean values accompanied by standard deviations. A significance threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied to ascertain statistical significance. Histopathological assessment of tissue samples from animals in Group \u0026lsquo;A\u0026rsquo; exhibited pronounced inflammatory manifestations within the fistulous tract, typified by abscess formation, necrotic tissue accumulation, and infiltration of immune cells. Comparative analysis revealed notably elevated pathology scores in Group \u0026lsquo;B\u0026rsquo; animals compared to those in Group \u0026lsquo;A\u0026rsquo;, characterized by a conspicuous presence of the fistulous tract and marked squamous cell epithelization, indicative of a more advanced pathological state.\u003c/p\u003e \u003cp\u003eThe histological images were analyzed for manual visual assessment of ulcers, lymphocytes, neutrophils, fibrosis, recessions and oedema. Blind analysis was performed by an expert on these slides. The ulcer was observed in both groups (20th and 40th day), but it was less pronounced in Group \u0026lsquo;B\u0026rsquo;, which could be due to recovery (granulation). The inflammatory cells (neutrophils and lymphocytes) and oedema were present in both groups without major differences. The chronic inflammatory response (fibrosis and recession) was much more pronounced in Group \u0026lsquo;B\u0026rsquo; compared to Group \u0026lsquo;A\u0026rsquo;. The classification can be seen in Table\u0026nbsp;12.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePerianal fistulas epitomize aberrant tracts or passages that develop in the perianal region, typically stemming from anal gland infection or inflammation. These pathological formations often manifest symptoms such as pain, swelling, and discharge around the anal area. Diagnosis commonly involves clinical evaluation by healthcare practitioners supplemented by imaging modalities such as MRI or ultrasound for comprehensive assessment. Treatment strategies vary based on severity and may encompass antibiotic therapy to address infection, coupled with surgical intervention to excise the fistulous tract and drain associated abscesses. In select cases, pharmacological agents, particularly immunosuppressants, may be prescribed, especially when underlying conditions like Crohn's disease are implicated. Despite therapeutic endeavors, perianal fistulas pose challenges due to their propensity for recurrence and potential complications such as chronic pain and fecal incontinence. Consequently, ongoing management requires vigilance, often necessitating long-term surveillance and continued medical intervention to optimize patient outcomes and mitigate recurrence rates.\u003c/p\u003e \u003cp\u003eThe management of \u003cem\u003eFistula-in-ano\u003c/em\u003e remains a persistent challenge for clinicians and surgeons alike, characterized by complexities in decision-making regarding surgical interventions, particularly with the advent of novel techniques in recent years [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Despite the efforts of experienced practitioners, achieving a consensus on optimal surgical approaches for \u003cem\u003eFistula-in-ano\u003c/em\u003e of cryptoglandular origin has proven elusive[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This discourse aims to explore the diverse trajectories of \u003cem\u003eFistula-in-ano\u003c/em\u003e, delineating their respective advantages and drawbacks. The rabbit emerges as a compelling choice for experimental and investigative endeavors in proctology, given its striking anatomical and histological resemblance to humans, as well as its manageable size and ease of manipulation. Our research endeavors to minimize animal distress while maximizing research outcomes through innovative methodologies. Notably, we present a novel approach to induce \u003cem\u003eFistula-in-ano\u003c/em\u003e in rabbits using solely SSSW No. 18G under general anesthesia, eschewing the use of chemical agents. Presently, no universally optimal curative treatment exists for this condition. In comparison to smaller animal models such as rats, rabbits, with their analogous anatomy to humans, offer a more suitable platform for studying \u003cem\u003eFistula-in-ano.\u003c/em\u003e Our investigation furnishes compelling morphological and histological evidence corroborating the resemblance between rabbit anatomy and that of humans, particularly concerning perineal and sphincter musculatures. The induction of rectal histological inflammation with perianal trans-sphincteric fistula formation was carefully observed through PUS and X-Ray Fistulogram [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Diagnosis primarily relied on PDAIS and histology, complemented by diagnostic modalities such as PUS and X-Ray Fistulogram. In this animal model, the induction of SSSW stimulates inflammation in the perianal region, initiating the wound healing cascade. Our approach, involved one time fistula induction using SSSW under general anesthesia, mimics human fistula recurrence while prolonging inflammation, thereby eliciting a robust inflammatory response. Notably, persistent inflammation induction led to improved disease scores in Group \u0026lsquo;B\u0026rsquo;, underscoring the significance of sustained stimulus for optimal fistula formation. Our findings demonstrate that a preclinical model of perianal fistula can be effectively established in rabbits with prolonged intact SSSW induction. Histopathological examination revealed features consistent with human perianal fistula, albeit further criteria validation is warranted. Despite the promising outcomes observed in our rabbit model, it is imperative to acknowledge the inherent limitations of this study. Unlike previous studies utilizing canine and porcine models, our approach showcased robust epithelialization and tissue formation post-fistula induction, suggesting the potential utility of the rabbit model in advancing our understanding and management of \u003cem\u003eFistula-in-ano.\u003c/em\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study conceals the development of a direct preclinical animal model that simulates perianal fistula formation in rabbits by using an extended technique to induce perianal fistula by prolonged (40 days) implantation of SSSW. This model is capable of mimicking the pathological condition in humans, whereby the induced fistula lesions can be assessed by PDAIS, histological analysis and additional diagnostic procedures such as PUS and X-ray examinations of the perianal region. The comprehensive evaluation carried out here should enrich research into innovative therapeutic strategies for the treatment of perianal fistula diseases.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGarcia-Aguilar, J., et al., \u003cem\u003eAnal fistula surgery: factors associated with recurrence and incontinence.\u003c/em\u003e 1996. \u003cstrong\u003e39\u003c/strong\u003e: p. 723-729.\u003c/li\u003e\n\u003cli\u003eSanchez-Haro, E., et al., \u003cem\u003eClinical characterization of patients with anal fistula during follow-up of anorectal abscess: a large population-based study.\u003c/em\u003e 2023. \u003cstrong\u003e27\u003c/strong\u003e(10): p. 897-907.\u003c/li\u003e\n\u003cli\u003eKrishnakanth, M., \u003cem\u003eEvaluation of Fistulectomy and Fistulotomy in the Management of Low Anal Fistula\u003c/em\u003e. 2018, Rajiv Gandhi University of Health Sciences (India).\u003c/li\u003e\n\u003cli\u003eHansdah, S. and T.J.A.o.t.R.S.f.C.B. Baxla, \u003cem\u003eThe Incidence of low Fistula in Ano and RESULTS of Fistulectomy.\u003c/em\u003e 2021. \u003cstrong\u003e25\u003c/strong\u003e(6): p. 6794-6797.\u003c/li\u003e\n\u003cli\u003eGarcia-Olmo, D., et al., \u003cem\u003ePrevalence of anal fistulas in Europe: systematic literature reviews and population-based database analysis.\u003c/em\u003e 2019. \u003cstrong\u003e36\u003c/strong\u003e(12): p. 3503-3518.\u003c/li\u003e\n\u003cli\u003eVogel, J.D., et al., \u003cem\u003eClinical practice guideline for the management of anorectal abscess, fistula-in-ano, and rectovaginal fistula.\u003c/em\u003e 2016. \u003cstrong\u003e59\u003c/strong\u003e(12): p. 1117-1133.\u003c/li\u003e\n\u003cli\u003eLu, S., et al., \u003cem\u003eEvaluation of animal models of Crohn\u0026apos;s disease with anal fistula.\u003c/em\u003e 2021. \u003cstrong\u003e22\u003c/strong\u003e(3): p. 1-7.\u003c/li\u003e\n\u003cli\u003eRafati, M., et al., \u003cem\u003eHuman amniotic membrane effect on perianal fistula healing in rabbits: an experimental study.\u003c/em\u003e 2018. \u003cstrong\u003e43\u003c/strong\u003e(5): p. 473.\u003c/li\u003e\n\u003cli\u003eWang, T., et al., \u003cem\u003eInjectable and self-healing microgels repairing infected irregular tubular fistula in anal and muscle.\u003c/em\u003e 2022. \u003cstrong\u003e441\u003c/strong\u003e: p. 135948.\u003c/li\u003e\n\u003cli\u003eSinger, M., et al., \u003cem\u003eTreatment of fistulas-in-ano with fibrin sealant in combination with intra-adhesive antibiotics and/or surgical closure of the internal fistula opening.\u003c/em\u003e 2005. \u003cstrong\u003e48\u003c/strong\u003e(4): p. 799-808.\u003c/li\u003e\n\u003cli\u003eFlacs, M., et al., \u003cem\u003ePreclinical model of perianal fistulizing Crohn\u0026rsquo;s disease.\u003c/em\u003e 2020. \u003cstrong\u003e26\u003c/strong\u003e(5): p. 687-696.\u003c/li\u003e\n\u003cli\u003eSingh, A., et al., \u003cem\u003eManagement of perianal fistulizing Crohn\u0026rsquo;s disease.\u003c/em\u003e 2023: p. izad195.\u003c/li\u003e\n\u003cli\u003eKural, T., \u003cem\u003eMicroscopic Structure of Intestinal Anastomoses and the Use of Animal Models in Experimental Intestinal Surgery.\u003c/em\u003e 2023.\u003c/li\u003e\n\u003cli\u003eBenlice, C., et al., \u003cem\u003eFistula tract curettage and the use of biological dermal plugs improve high transsphincteric fistula healing in an animal model.\u003c/em\u003e 2016. \u003cstrong\u003e31\u003c/strong\u003e: p. 291-299.\u003c/li\u003e\n\u003cli\u003eDe la Portilla, F., et al., \u003cem\u003eThe rabbit as an animal model for proctology research: anatomical and histological description.\u003c/em\u003e 2011. \u003cstrong\u003e24\u003c/strong\u003e(3): p. 134-137.\u003c/li\u003e\n\u003cli\u003eLosco, A., et al., \u003cem\u003eAssessing the activity of perianal Crohn\u0026apos;s disease: comparison of clinical indices and computer-assisted anal ultrasound.\u003c/em\u003e Inflamm Bowel Dis, 2009. \u003cstrong\u003e15\u003c/strong\u003e(5): p. 742-9.\u003c/li\u003e\n\u003cli\u003eGecse, K.B., et al., \u003cem\u003eResults of the Fifth Scientific Workshop of the ECCO [II]: Clinical Aspects of Perianal Fistulising Crohn\u0026rsquo;s Disease\u0026mdash;the Unmet Needs.\u003c/em\u003e Journal of Crohn\u0026apos;s and Colitis, 2016. \u003cstrong\u003e10\u003c/strong\u003e(7): p. 758-765.\u003c/li\u003e\n\u003cli\u003eAkhoundi, N., et al., \u003cem\u003eComparison of MRI and Endoanal Ultrasound in Assessing Intersphincteric, Transsphincteric, and Suprasphincteric Perianal Fistula.\u003c/em\u003e J Ultrasound Med, 2023. \u003cstrong\u003e42\u003c/strong\u003e(9): p. 2057-2064.\u003c/li\u003e\n\u003cli\u003eMacPherson, B.R. and C.J. Pfeiffer, \u003cem\u003eExperimental production of diffuse colitis in rats.\u003c/em\u003e Digestion, 1978. \u003cstrong\u003e17\u003c/strong\u003e(2): p. 135-50.\u003c/li\u003e\n\u003cli\u003eKontovounisios, C., et al., \u003cem\u003eAdoption and success rates of perineal procedures for fistula‐in‐ano: a systematic review.\u003c/em\u003e 2016. \u003cstrong\u003e18\u003c/strong\u003e(5): p. 441-458.\u003c/li\u003e\n\u003cli\u003eDeeba, S., et al., \u003cem\u003eFistula-in-ano: advances in treatment.\u003c/em\u003e 2008. \u003cstrong\u003e196\u003c/strong\u003e(1): p. 95-99.\u003c/li\u003e\n\u003cli\u003eJi, L., et al., \u003cem\u003eAdvances in the treatment of anal fistula: a mini-review of recent five-year clinical studies.\u003c/em\u003e 2021. \u003cstrong\u003e7\u003c/strong\u003e: p. 586891.\u003c/li\u003e\n\u003cli\u003eShahzad, M., et al., \u003cem\u003eEffectiveness of radiological imaging techniques (x-rays, mdct, and mri) for diagnosis of pelvic fistula: A systematic review.\u003c/em\u003e 2021. \u003cstrong\u003e2021\u003c/strong\u003e(1).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 12 are available in the Supplementary Files section.\u003c/p\u003e "},{"header":"Annexure ","content":"\u003cp\u003eAnnexure 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Government Nizamia Tibbia College, Charminar, Hyderabad T.S","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Induced Fistula-in-ano, Nassor-e-Maqad, Rabbit Model, Perianal Fistula, Experimental Study","lastPublishedDoi":"10.21203/rs.3.rs-4144162/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4144162/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eFistula-in-ano\u003c/em\u003e, termed ‘\u003cem\u003eNassor-e-Maqad’\u003c/em\u003ein traditional Arabic medicine, presents a challenging perianal pathology necessitating a robust experimental framework for comprehensive inquiry. This investigation endeavors to establish an induced \u003cem\u003eFistula-in-ano\u003c/em\u003e model in New Zealand White (NZW) Rabbits (n=12) utilizing a protocol involving surgical suturing steel wire implantation for durations of 20 days (Group ‘A’) and 40 days (Group ‘B').\u003c/p\u003e\n\u003cp\u003eThe principal aim of this research is to develop an induced \u003cem\u003eFistula-in-ano\u003c/em\u003emodel in NZW rabbits, with the objective of enhancing comprehension of perianal disease pathogenesis and exploring novel therapeutic modalities. Given the absence of universally efficacious and complication-free treatments, the development of innovative therapeutic approaches is imperative. Hence, the creation of an optimal animal model capable of simulating persistent anal fistulas with lesions resembling clinical manifestations is vital for preclinical investigations in this domain.\u003c/p\u003e\n\u003cp\u003eUpon imaging (X-ray and perianal ultrasonography) the patency of fistula was evaluated whereas histological evaluation exhibited that animals in Group ‘A’ (n=06) fistula has openings with modest epithelialization subsequent to the removal of the surgical steel suture ring, along with favorable diagnostic outcomes, 20 days post-surgery. Conversely, animals in Group ‘B’ (n=6), upon steel wire removal after 40 days, displayed notable changes in epithelization, indicative of further progression in the induced fistula model.\u003c/p\u003e\n\u003cp\u003eIn summary, this study successfully establishes a preclinical rabbit model of perianal fistula devoid of chemical agents, relying solely on surgical induction \u003cem\u003evia\u003c/em\u003e surgical suturing steel wire (SSSW). The considerable anatomical and histological parallels between rabbit and human perianal pathophysiology underscore the suitability of rabbits as an experimental model for advancing therapeutic strategies in proctology.\u003c/p\u003e","manuscriptTitle":"Bridging the Research Gap: Establishing a Rabbit Model for Perianal Fistula (Nassor-E Maqad) Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-26 18:11:42","doi":"10.21203/rs.3.rs-4144162/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"44ea1cc5-5a12-4055-82a8-9783f7d46006","owner":[],"postedDate":"March 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29727128,"name":"Animal Science"},{"id":29727129,"name":"Gastrointestinal Surgery"}],"tags":[],"updatedAt":"2024-03-26T18:11:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-26 18:11:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4144162","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4144162","identity":"rs-4144162","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}